SMART MODE:

NORMAL GENOMIC

• Merriweather A, Guenzler V, Brenner M, Unnasch TR
• Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase.
• Mol Biochem Parasitol. 2001; 116: 185-97
• Display abstract

• The cuticle of parasitic nematodes consists primarily of a network of collagen molecules. The enzyme responsible for collagen maturation is prolyl 4-hydroxylase, making this enzyme a central activity in cuticle biosynthesis and a potentially important chemotherapeutic target. Adult and embryonic Brugia malayi are shown to be susceptible to inhibitors of vertebrate prolyl 4-hydroxylase, with exposed parasites exhibiting pathologies consistent with a disruption in cuticle biosynthesis. A full-length cDNA (Ov-phy-1) encoding a catalytically active alpha-subunit of Onchocerca volvulus prolyl 4-hydroxylase was isolated and characterized. The derived amino acid sequence of Ov-phy-1 encoded a peptide that was most similar to the two Caenorhabditis elegans prolyl 4-hydroxylase homologues and to the isoform II enzymes of vertebrates. Expressed sequence tag (EST) analysis and developmental polymerase chain reaction (PCR) studies demonstrated that Ov-phy-1 was expressed in L3 and adult parasites. The gene encoding the Ov-phy-1 open reading frame contained 11 introns, similar in structure to the gene encoding human prolyl 4-hydroxylase isoform I. Genomic Southern blot, EST and genomic PCR studies demonstrated that the O. volvulus genome contained between three and eight genes closely related to Ov-phy-1. Co-expression of Ov-phy-1 with the O. volvulus homologue of protein disulfide isomerase in a baculovirus system resulted in the production of enzymatically active O. volvulus prolyl 4-hydroxylase. In vitro production of enzymatically active O. volvulus prolyl 4-hydroxylase should facilitate identification of specific inhibitors of the parasite enzyme.

• Franklin TJ, Morris WP, Edwards PN, Large MS, Stephenson R
• Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones.
• Biochem J. 2001; 353: 333-8
• Display abstract

• Examples of a novel series of phenanthrolinones are shown to be potent competitive inhibitors of avian prolyl 4-hydroxylase, and of collagen hydroxylation, in embryonic chick tendon cells and human foreskin fibroblasts in vitro and in the oestradiol-stimulated rat uterus in vivo. Two compounds, Compound 1 (1,4-dihydrophenanthrolin-4-one-3-carboxylic acid) and Compound 5 [8-(N-butyl-N-ethylcarbamoyl)-1,4-dihydrophenathrolin-4-one-3-carboxylic acid], with comparable potencies in vivo, were chosen to investigate the effect of the inhibition of the hydroxylation of newly synthesized uterine collagen on the turnover of this protein in vivo. Inhibition of hydroxylation by more than 50% for approx. 8 h following single oral doses of the compounds was associated with significant losses of radiolabelled proline and 4-hydroxyproline from collagen during this period. Progressive hydroxylation of collagen over 48 h, as the inhibitory action of the compounds declined, was accompanied by a decreased loss of radiolabel from the uterine collagen. Earlier reports indicated that underhydroxylated collagen, accumulating within the endoplasmic reticulum in cells where prolyl 4-hydroxylase is inactivated, is slowly degraded, but is then rapidly hydroxylated and secreted when the activity of prolyl 4-hydroxylase is restored. Taken with the present results, this suggests that the potential use of inhibitors of prolyl 4-hydroxylase to control excessive collagen deposition in pathological fibrosis may be limited by the need to maintain continuous inhibition of collagen hydroxylation so as to facilitate intracellular degradation of the accumulated protein.

• Lee EH, Kao WW, Schwarz RI
• Cell density regulates prolyl 4-hydroxylase activity independent of mRNA levels.
• Matrix Biol. 2001; 19: 779-82
• Display abstract

• In embryonic avian tendon, cell density regulates collagen production. This control is propagated through the alpha-subunit of prolyl 4-hydroxylase where protein levels were previously shown to rise fivefold with increasing cell density. In contrast, mRNA levels are now shown not to change by both Northern and RNAse protection assays. This lack of increase contrasts with previous reports as does the mRNA length: this is 50% larger as confirmed by sequencing the 3' end. Alternative sites for cell density regulation of the enzyme could rely on its sensitivity to sulfhydryl groups. Using a fluorescent sulfhydryl probe as well as a sulfhydryl inhibitor, one observes a strong cell density response, supporting the hypothesis that cellular redox potential could alter protein stability.

• Pirneskoski A, Ruddock LW, Klappa P, Freedman RB, Kivirikko KI, Koivunen P
• Domains b' and a' of protein disulfide isomerase fulfill the minimum requirement for function as a subunit of prolyl 4-hydroxylase. The N-terminal domains a and b enhances this function and can be substituted in part by those of ERp57.
• J Biol Chem. 2001; 276: 11287-93
• Display abstract

• Protein disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a', plus an acidic C-terminal extension, c. PDI carries out multiple functions, acting as the beta subunit in the animal prolyl 4-hydroxylases and in the microsomal triglyceride transfer protein and independently acting as a protein folding catalyst. We report here that the minimum sequence requirement for the assembly of an active prolyl 4-hydroxylase alpha(2)beta(2) tetramer in insect cell coexpression experiments is fulfilled by the PDI domain construct b'a' but that the sequential addition of the b and a domains greatly increases the level of enzyme activity obtained. In the assembly of active prolyl 4-hydroxylase tetramers, the a and b domains of PDI, but not b' and a', can in part be substituted by the corresponding domains of ERp57, a PDI isoform that functions naturally in association with the lectins calnexin and calreticulin. The a' domain of PDI could not be substituted by the PDI a domain, suggesting that both b' and a' domains contain regions critical for prolyl 4-hydroxylase assembly. All PDI domain constructs and PDI/ERp57 hybrids that contain the b' domain can bind the 14-amino acid peptide Delta-somatostatin, as measured by cross-linking; however, binding of the misfolded protein "scrambled" RNase required the addition of domains ab or a' of PDI. The human prolyl 4-hydroxylase alpha subunit has at least two isoforms, alpha(I) and alpha(II), which form with the PDI polypeptide the (alpha(I))(2)beta(2) and (alpha(II))(2)beta(2) tetramers. We report here that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides tested were more effectively associated with the alpha(II) subunit than the alpha(I) subunit.

• Kim HS, Colgan SP, Pitman R, Hershberg RM, Blumberg RS
• Human CD1d associates with prolyl-4-hydroxylase during its biosynthesis.
• Mol Immunol. 2000; 37: 861-8
• Display abstract

• Recent studies have shown that the CD1 family of proteins present various glycolipid antigens to subsets of T cells. CD1d is expressed on human intestinal epithelial cells (IEC) and exists in two biochemical forms: 37-kDa, beta2-microglobulin (beta2m) independent, nonglycosylated, and 47-kDa, beta2m dependent, glycosylated forms. The biosynthetic pathways and the mechanisms of generation of these two biochemically distinct forms of CD1d in human IEC are unknown. Using a human colonic cell line, T84, transfected with CD1d, the biosynthesis of CD1d was investigated. Pulse-chase metabolic labeling studies of T84 transfected with wild type CD1d demonstrated that CD1d was a stable protein over a 4-day chase period. During the first 24 h of the chase, a novel 65-kDa glycoprotein was co-immunoprecipitated with CD1d. Microsequencing of this protein identified the glycoprotein as the alpha and beta subunits of the resident endoplasmic reticulum protein, prolyl-4-hydroxylase (P4H), an enzyme responsible for hydroxyl modification of proline residues. To study if either one or both biochemical forms of CD1d contained hydroxyproline residues, amino acid composition analysis of the 37 and 48 kDa was performed, and demonstrated that only the 37-kDa, but not the 48-kDa form of CD1d, contained hydroxyproline residues. These studies demonstrate that CD1d exhibits a prolonged association with P4H and that the 37-kDa form contains hydroxyproline residues. This suggests that P4H association with CD1d during its biosynthesis results in a novel post-translational modification of CD1d.

• Wang C, Valtavaara M, Myllyla R
• Lack of collagen type specificity for lysyl hydroxylase isoforms.
• DNA Cell Biol. 2000; 19: 71-7
• Display abstract

• Lysyl hydroxylase is the enzyme catalyzing the formation of hydroxylysyl residues in collagens. Large differences in the extent of hydroxylysyl residues are found among collagen types. Three lysyl hydroxylase isoenzymes (LH1, LH2, LH3) have recently been characterized from human and mouse tissues. Nothing is known about the distribution of these isoforms within cells or whether they exhibit collagen type specificity. We measured mRNA levels of the three isoforms, as well as the mRNAs of the main collagen types I, III, IV, and V and the alpha subunit of prolyl 4-hydroxylase, another enzyme involved in collagen biosynthesis, in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. Immunoblotting was utilized to confirm the results of Northern hybridization. The levels of mRNA of LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase showed significant correlations with each other. The LH3 mRNA levels did not correlate with those of LH1, LH2, or the alpa subunit of prolyl 4-hydroxylase, clearly indicating a difference in the regulation of LH3. No correlation was observed between LH isoforms and individual collagen types, indicating a lack of collagen type specificity for lysyl hydroxylase isoforms. Our observations suggest that LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase are coregulated together with total collagen synthesis but not with the specific collagen types and indicate that LH3 behaves differently from LH1 and LH2, implying a difference in their substrates. These observations set the basis for further studies to define the functions of lysyl hydroxylase isoforms.

• Hosokawa N, Nagata K
• Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate.
• FEBS Lett. 2000; 466: 19-25
• Display abstract

• In cells, only properly folded procollagen trimers are secreted from the endoplasmic reticulum (ER), while improperly folded abnormal procollagens are retained within the ER. Ascorbic acid is a co-factor in procollagen hydroxylation, which in turn is required for trimer formation. We examined chaperone proteins which bound to procollagen in the absence of ascorbic acid, a model which mimics the human disease scurvy at the cellular level. We found that both prolyl 4-hydroxylase (P4-H)/protein disulfide isomerase (PDI) and HSP47 bound to procollagen in the absence of ascorbic acid. However, the binding of PDI to procollagen decreased when HSP47 was co-transfected, suggesting that HSP47 and PDI compete for binding to procollagen. These data indicate that P4-H/PDI and HSP47 have cooperative but distinct chaperone functions during procollagen biosynthesis.

• Remm M, Sonnhammer E
• Classification of transmembrane protein families in the Caenorhabditis elegans genome and identification of human orthologs.
• Genome Res. 2000; 10: 1679-89
• Display abstract

• The complete genome sequence of the nematode Caenorhabditis elegans provides an excellent basis for studying the distribution and evolution of protein families in higher eukaryotes. Three fundamental questions are as follows: How many paralog clusters exist in one species, how many of these are shared with other species, and how many proteins can be assigned a functional counterpart in other species? We have addressed these questions in a detailed study of predicted membrane proteins in C. elegans and their mammalian homologs. All worm proteins predicted to contain at least two transmembrane segments were clustered on the basis of sequence similarity. This resulted in 189 groups with two or more sequences, containing, in total, 2647 worm proteins. Hidden Markov models (HMMs) were created for each family, and were used to retrieve mammalian homologs from the SWISSPROT, TREMBL, and VTS databases. About one-half of these clusters had mammalian homologs. Putative worm-mammalian orthologs were extracted by use of nine different phylogenetic methods and BLAST. Eight clusters initially thought to be worm-specific were assigned mammalian homologs after searching EST and genomic sequences. A compilation of 174 orthology assignments made with high confidence is presented.

• Takahashi Y, Takahashi S, Shiga Y, Yoshimi T, Miura T
• Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells.
• J Biol Chem. 2000; 275: 14139-46
• Display abstract

• Accumulated evidence indicates that hypoxia activates collagen synthesis in tissues. To explore the molecular mechanism of activation, we screened genes that are up-regulated or down-regulated by hypoxia. Fibroblasts isolated from fetal rat lung were cultured under hypoxia. Differential display technique showed that the mRNA level of prolyl 4-hydroxylase (PH) alpha(I), an active subunit that catalyzes the oxygen-dependent hydroxylation of proline residue in procollagen, increased 2-3-fold after an 8-h exposure to hypoxia. This elevated level was maintained over 40 h and returned to the basal level after reoxygenation. The transcription rate, protein level, and hydroxyproline content (an indicator of the prolyl hydroxylation) were all elevated by hypoxic culture. Analysis of the promotor region of PHalpha(I) gene indicated that a motif similar to hypoxia-responsive element (HRE) of hypoxia-inducible genes such as erythropoietin, was identified within a 120-base pair sequence upstream of the transcription start site. Luciferase reporter assay and mutational analysis showed that a site similar to the HRE in this motif is functionally essential to hypoxic response. Electrophoretic mobility shift assay revealed that hypoxia-inducible factor-1 was stimulated and bound to the PHalpha(I) HRE upon hypoxic challenge. Our results indicate that PHalpha(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.

• Winter AD, Page AP
• Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caenorhabditis elegans.
• Mol Cell Biol. 2000; 20: 4084-93
• Display abstract

• The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The beta subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase alpha-subunit (PHY; EC 1.14.11.2)- and beta-subunit (PDI; EC 5.3.4.1)-encoding genes with respect to their role in collagen modification and formation of the C. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combined phy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterized dpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.

• Wu M, Moon HS, Pirskanen A, Myllyharju J, Kivirikko KI, Begley TP
• Mechanistic studies on prolyl-4-hydroxylase: the vitamin C requiring uncoupled oxidation.
• Bioorg Med Chem Lett. 2000; 10: 1511-4
• Display abstract

• A deuterated substrate for the human type I prolyl-4-hydroxylase was synthesized and its V/K deuterium isotope effect was determined to be 3.4 +/- 0.2. This isotope effect was attributed to the uncoupled oxidation. A dehydroproline containing tetrapeptide was also found to stimulate the uncoupled oxidation.

• Myllyharju J, Nokelainen M, Vuorela A, Kivirikko KI
• Expression of recombinant human type I-III collagens in the yeast pichia pastoris.
• Biochem Soc Trans. 2000; 28: 353-7
• Display abstract

• An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and-III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.

• Kaapa E et al.
• Elevated protein content and prolyl 4-hydroxylase activity in severely degenerated human annulus fibrosus.
• Connect Tissue Res. 2000; 41: 93-9
• Display abstract

• Alterations involved with the intervertebral disc degeneration are partly well described, however, it is not so well known how collagen network is affected by the disease. We analyzed the rate of collagen biosynthesis (estimated by the enzymic activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase) and the level of hydroxylysylpyridinoline and lysylpyridinoline crosslinks both in normal (n=7) and degenerated (n=7) human annulus fibrosus. The activity of prolyl 4-hydroxylase was significantly increased in degenerated tissue. However, no significant changes in the collagen content or in the amount of hydroxylysylpyridinoline and lysylpyridinoline collagen crosslinks were observed. On the other hand, the content of soluble proteins was significantly increased. Our results suggest that collagen biosynthesis is increased in degenerated human annulus fibrosus, obviously to compensate the impairment of collagen fibers. The faster turnover of collagen in degenerated annulus fibrosus, suggested by the increased prolyl 4-hydroxylase activity and unchanged collagen content, seems not to cause any significant changes in its mature pyridinium crosslink concentrations.

• Friedman L, Higgin JJ, Moulder G, Barstead R, Raines RT, Kimble J
• Prolyl 4-hydroxylase is required for viability and morphogenesis in Caenorhabditis elegans.
• Proc Natl Acad Sci U S A. 2000; 97: 4736-41
• Display abstract

• The genome of Caenorhabditis elegans possesses two genes, dpy-18 and phy-2, that encode alpha subunits of the enzyme prolyl 4-hydroxylase. We have generated deletions within each gene to eliminate prolyl 4-hydroxylase activity from the animal. The dpy-18 mutant has an aberrant body morphology, consistent with a role of prolyl 4-hydroxylase in formation of the body cuticle. The phy-2 mutant is phenotypically wild type. However, the dpy-18; phy-2 double mutant is not viable, suggesting an essential role for prolyl 4-hydroxylase that is normally accomplished by either dpy-18 or phy-2. The effects of the double mutation were mimicked by small-molecule inhibitors of prolyl 4-hydroxylase, validating the genetic results and suggesting that C. elegans can serve as a model system for the discovery of new inhibitors.

• Yamada S, Kawasaki H
• [Prolyl hydroxylase (PH)]
• Nippon Rinsho. 1999; 57: 422-4

• John DC, Watson R, Kind AJ, Scott AR, Kadler KE, Bulleid NJ
• Expression of an engineered form of recombinant procollagen in mouse milk.
• Nat Biotechnol. 1999; 17: 385-9
• Display abstract

• We have examined the suitability of the mouse mammary gland for expression of novel recombinant procollagens that can be used for biomedical applications. We generated transgenic mouse lines containing cDNA constructs encoding recombinant procollagen, along with the alpha and beta subunits of prolyl 4-hydroxylase, an enzyme that modifies the collagen into a form that is stable at body temperature. The lines expressed relatively high levels (50-200 micrograms/ml) of recombinant procollagen in milk. As engineered, the recombinant procollagen was shortened and consisted of a pro alpha 2(I) chain capable of forming a triple-helical homotrimer not normally found in nature. Analysis of the product demonstrated that (1) the pro alpha chains formed disulphide-linked trimers, (2) the trimers contained a thermostable triple-helical domain, (3) the N-propeptides were aligned correctly, and (4) the expressed procollagen was not proteolytically processed to collagen in milk.

• Eriksson M, Myllyharju J, Tu H, Hellman M, Kivirikko KI
• Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates.
• J Biol Chem. 1999; 274: 22131-4
• Display abstract

• 4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins. We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase.

• Wojtaszek P, Smith CG, Bolwell GP
• Ultrastructural localisation and further biochemical characterisation of prolyl 4-hydroxylase from Phaseolus vulgaris: comparative analysis.
• Int J Biochem Cell Biol. 1999; 31: 463-77
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2), the enzyme responsible for the post-translational hydroxylation of peptide proline, has been well described in animals but has been little studied in plants. The best characterised example is the enzyme from French bean (Phaseolus vulgaris). In this study, the biochemical properties of this plant enzyme were examined in more detail and, using specific polyclonal antibodies, the localisation of the enzyme was determined. Both alpha- and beta-subunits did not show multiple forms, suggesting a relatively broad specificity of the enzyme complex with respect to the target hydroxylated amino acid sequences. Antibodies to the mammalian and Chlamydomonas enzymes cross-react with the higher plant subunits, indicating that some epitopes are highly conserved. The P. vulgaris enzyme was inhibited by analogues of oxoglutarate, but was not susceptible to doxorubicin. Inhibition of the bean enzyme by an oxaloglycine derivative resulted in the retention of the target (hydroxy)proline-rich protein in the endomembrane system. Immunolocalisation of the enzyme showed close association with the endoplasmic reticulum and Golgi apparatus in root tip cells of P. vulgaris or Tropaeolum majus. This localisation was particularly pronounced in Golgi-associated vesicles of young root tip cells of T. majus, cell types where rapid synthesis and deposition of wall material was observed. These data are consistent with the hypothesis, proposed by Bolwell [G.P. Bolwell, Dynamic aspects of the plant extracellular matrix, Int. Rev. Cytol. 146 (1993) 261-324], that protein hydroxylation must be completed before the glycosylation of the target (hydroxy)proline-rich glycoproteins in the Golgi stack.

• Walmsley AR, Batten MR, Lad U, Bulleid NJ
• Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.
• J Biol Chem. 1999; 274: 14884-92
• Display abstract

• The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly.

• Annunen P, Koivunen P, Kivirikko KI
• Cloning of the alpha subunit of prolyl 4-hydroxylase from Drosophila and expression and characterization of the corresponding enzyme tetramer with some unique properties.
• J Biol Chem. 1999; 274: 6790-6
• Display abstract

• Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, whereas the Caenorhabditis elegans enzyme is an alphabeta dimer, the beta subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha subunit and 31% identity to the C. elegans alpha subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1. 9-kilobase mRNA coding for this alpha subunit was present in Drosophila larvae.

• Myllyharju J, Kivirikko KI
• Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.
• EMBO J. 1999; 18: 306-12
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase

• Vuorela A, Myllyharju J, Pihlajaniemi T, Kivirikko KI
• Coexpression with collagen markedly increases the half-life of the recombinant human prolyl 4-hydroxylase tetramer in the yeast Pichia pastoris.
• Matrix Biol. 1999; 18: 519-22
• Display abstract

• Recent coexpression studies of the subunits of human prolyl 4-hydroxylase (4-PH) in the yeast Pichia pastoris have indicated that only a minor fraction of them were present in the alpha2beta2 tetramer, while coexpression with type III procollagen markedly increased their assembly level. We report here that the half-life of the recombinant 4-PH tetramer in Pichia when studied by pulse-chase experiments was only 50 min. Coexpression with the pro alpha1(III) chains increased this half-life to 12.5 h. Coexpression with the pro alpha1(I) chains, which were produced at half the level of the pro alpha1(III) chains, gave a half-life of 6.5 h. Coexpression with collagen thus markedly increases the half-life of the 4-PH tetramer, and the half-life may be related to the level of collagen expression.

• Asada S, Koide T, Yasui H, Nagata K
• Effect of HSP47 on prolyl 4-hydroxylation of collagen model peptides.
• Cell Struct Funct. 1999; 24: 187-96
• Display abstract

• Prolyl 4-hydroxylation, the most important post-translational modification in collagen biosynthesis, is catalyzed by prolyl 4-hydroxylase, an endoplasmic reticulum-resident enzyme. HSP47 is a collagen-binding stress protein which also resides in the endoplasmic reticulum (Nagata, K. and Yamada, K.M. (1986) J. Biol. Chem., 261, 7531-7536). Both prolyl 4-hydroxylase and HSP47 interact with procollagen alpha-chains during their folding and/or modification in the endoplasmic reticulum. Recent study has revealed that a simple collagen model peptide, (Pro-Pro-Gly)n, is recognized by HSP47 as well as by prolyl 4-hydroxylase in vitro (Koide et al., manuscript submitted). In the present study, we investigated the effect of HSP47 on the prolyl 4-hydroxylation of such collagen model peptides. To monitor the enzymatic hydroxylation of the peptides, we developed a non-RI assay system based on reversed-phase HPLC. When HSP47 was added to the reaction mixture, substrate and less-hydroxylated materials accumulated. This effect depended on the peptide-binding activity of HSP47, because a mutant HSP47 without collagen-binding activity did not show any inhibitory effect on prolyl 4-hydroxylation. Kinetic analysis and other biochemical analyses suggest that HSP47 retards the enzymatic reaction competing for the substrate peptide.

• Koide T, Asada S, Nagata K
• Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation.
• J Biol Chem. 1999; 274: 34523-6
• Display abstract

• Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly)(n), possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly)(n) has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-Pro-Gly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly)(n), does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.

• Kivirikko KI, Pihlajaniemi T
• Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases.
• Adv Enzymol Relat Areas Mol Biol. 1998; 72: 325-98
• Display abstract

• Prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in collagens and other proteins with an appropriate collagen-like stretch of amino acid residues. The enzyme requires Fe(II), 2-oxoglutarate, molecular oxygen, and ascorbate. This review concentrates on recent progress toward understanding the detailed mechanism of 4-hydroxylase action, including: (a) occurrence and function of the enzyme in animals; (b) general molecular properties; (c) intracellular sites of hydroxylation; (d) peptide substrates and mechanistic roles of the cosubstrates; (e) insights into the development of antifibrotic drugs; (f) studies of the enzyme's subunits and their catalytic function; and (g) mutations that lead to Ehlers-Danlos Syndrome. An account of the regulation of collagen hydroxylase activities is also provided.

• Kaule G, Timpl R, Gaill F, Gunzler V
• Prolyl hydroxylase activity in tissue homogenates of annelids from deep sea hydrothermal vents.
• Matrix Biol. 1998; 17: 205-12
• Display abstract

• Tissue homogenates of the deep sea annelids Alvinella caudata and Alvinella pompejana were found to contain enzyme activity resembling vertebrate prolyl 4-hydroxylase. The release of 3H2O from [3,4-(3)H]proline labeled, under-hydroxylated chicken protocollagen type I depended on the presence of the cofactors 2-oxoglutarate, ascorbate, Fe2+ and O2. The release of 3H2O could be inhibited by the prolyl 4-hydroxylase inhibitors zinc, 2,2'-dipyridyl, 3,4-dihydroxybenzoic acid and pyridine-2,4-dicarboxylate, as well as by the synthetic peptide (Pro-Pro-Gly)10. This synthetic peptide could also serve as substrate, because it enhanced the decarboxylation of 2-oxo[5-(14)C]glutarate. Alvinella prolyl hydroxylase appeared to be related to type II vertebrate enzyme because of its lack of affinity for poly (L-proline) and resistance to inactivation by an irreversible peptide inhibitor of chicken prolyl 4-hydroxylase. Maximal enzyme activity was observed in solutions with less than 10% oxygen saturation. By contrast, chicken enzyme was most active at saturating oxygen concentrations. Further data suggest that the Alvinella enzymes are able to accept the 2-oxo acids pyruvate, oxaloacetate and 2-oxoadipinate as substitutes of the cosubstarate 2-oxoglutarate. The data explain the high hydroxylation of Alvinella collagens despite the low oxygen concentrations around hydrothermal vents.

• Annunen P, Autio-Harmainen H, Kivirikko KI
• The novel type II prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells.
• J Biol Chem. 1998; 273: 5989-92
• Display abstract

• Procollagen-proline dioxygenase (EC 1.14.11.2), an alpha2beta2 tetramer in vertebrates, plays a central role in the synthesis of all collagens. Recently an isoform of the alpha subunit, the alpha(II) subunit, was characterized in man and mouse and found to form a tetramer with the same beta subunit as the previously known alpha(I) subunit. We report here that the (alpha(I))2beta2 type I tetramer is the main enzyme form in most cell types and tissues and that its contribution to total prolyl 4-hydroxylase activity in cultured cells increases in confluence. Surprisingly, however, the (alpha(II))2beta2 type II enzyme was found to represent at least about 70% of the total prolyl 4-hydroxylase activity in cultured mouse chondrocytes and about 80% in mouse cartilage, the corresponding percentage in mouse bone being about 45% and that in many other mouse tissues about 10% or less. Immunofluorescence studies on samples from a fetal human foot confirmed these data and additionally indicated that the type II enzyme represents the main or only enzyme form in capillary endothelial cells. Thus the type II prolyl 4-hydroxylase is likely to play a major role in the development of cartilages and cartilaginous bones and also of capillaries.

• Tandon M, Wu M, Begley TP, Myllyharju J, Pirskanen A, Kivirikko K
• Substrate specificity of human prolyl-4-hydroxylase.
• Bioorg Med Chem Lett. 1998; 8: 1139-44
• Display abstract

• Proline analogs (3-F, 3-Cl, 3-Br, 3,3-cyclopropyl, 3,3-methylene, 3-Me, and 4-Me) were synthesized, incorporated into CbzGlyPheXGlyOEt, and tested as substrate analogues/mechanistic probes for the human prolyl-4-hydroxylase catalyzed hydroxylation reaction. With the exception of the 3-fluoroproline containing peptide, none of these peptides were substrates for the enzyme.

• Kivirikko KI, Myllyharju J
• Prolyl 4-hydroxylases and their protein disulfide isomerase subunit.
• Matrix Biol. 1998; 16: 357-68
• Display abstract

• Prolyl 4-hydroxylases (EC 1.14,11.2) catalyze the formation of 4-hydroxyproline in collagens and other proteins with collagen-like sequences. The vertebrate type I and type II enzymes are [alpha (I)]2 beta 2 and [alpha (II)]2 beta 2 tetramers, respectively, whereas the enzyme from the nematode Caenorhabditis elegans is an alpha beta dimer. The type I enzyme is the major form in most but not all vertebrate tissues. The catalytic properties of the various enzyme forms are highly similar, but there are distinct, although small, differences in K(m) values for various peptide substrates between the enzyme forms and major differences in Ki values for the competitive inhibitor, poly(L-proline). Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. Kinetic studies and theoretical considerations have led to elucidation of the reaction mechanism, and recent extensive site-directed mutagenesis studies have identified five critical residues at the cosubstrate binding sites. A number of compounds have been characterized that inhibit it competitively with respect to some of the cosubstrates, and three groups of suicide inactivators have also been identified. The beta subunit in all forms of prolyl 4-hydroxylase is identical to protein disulfide isomerase (PDI), a multifunctional polypeptide that also serves as a subunit in the microsomal triglyceride transfer protein, as a chaperone-like polypeptide that probably assists folding of a number of newly synthesized proteins, and in several other functions. The main role of the PDI polypeptide as a protein subunit is probably related to its chaperone function. Recent expression studies of recombinant human prolyl 4-hydroxylase subunits in a yeast have indicated that the formation of a stable enzyme tetramer in vivo requires coexpression of collagen polypeptide chains.

• Ju H, Hao J, Zhao S, Dixon IM
• Antiproliferative and antifibrotic effects of mimosine on adult cardiac fibroblasts.
• Biochim Biophys Acta. 1998; 1448: 51-60
• Display abstract

• Prolyl 4-hydroxylase catalyzes the hydroxylation of collagen pro-alpha chains for the deposition of cardiac collagen. The effect of prolyl 4-hydroxylase on synthesis and degradation of collagen was studied in cultured adult cardiac fibroblasts using mimosine, a prolyl 4-hydroxylase inhibitor. Mimosine inhibited [3H]thymidine incorporation in cultured fibroblasts in a dose-dependent manner (100-600 microM). Immunofluorescence in fibroblasts and biochemical detection of mature type I collagen in culture serum revealed a strong inhibition of synthesis and secretion of mature collagens, respectively, in the presence of 200 microM mimosine. Western blot analysis for procollagen was carried out in cultured fibroblasts, and 200 microM mimosine treatment was associated with increased intracellular accumulation of procollagen from 4.14+/-0.27 to 10. 19+/-0.37 (arbitrary units). Immunofluorescence studies confirmed a marked increase of intracellular procollagens in fibroblasts treated with mimosine, which suggests a loss of coordinated monomeric procollagen synthesis and secretion of triple helical mature collagens. Modest inhibition of collagen type I mRNA abundance was observed in mimosine-treated fibroblasts, whereas no effect was noted for mRNAs of collagen type III, alpha-prolyl 4-hydroxylase or beta-prolyl 4-hydroxylase when compared to untreated control values. Treatment of fibroblasts with 200 microM mimosine was associated with elevation of matrix metalloproteinase (MMP)-9 activity. The cytotoxicity of mimosine treatment was found minimal at the concentrations indicated above. Thus the antifibrotic effects induced by mimosine on cultured adult cardiac fibroblasts was associated with inhibition of prolyl 4-hydroxylase and diminished extracellular secretion of procollagen, despite the reactive elevation of intracellular procollagen synthesis. We suggest that specific inhibition of prolyl 4-hydroxylase may provide a novel therapeutic approach for the modulation of cardiac fibrosis.

• Saika S et al.
• Immunolocalization of prolyl 4-hydroxylase in rabbit lens epithelial cells.
• J Cataract Refract Surg. 1998; 24: 1261-5
• Display abstract

• PURPOSE: To localize the enzyme prolyl 4-hydroxylase in the crystalline lens and determine the ability of lens epithelial cells (LECs) to synthesize procollagen. SETTING: Research laboratory, Department of Ophthalmology, Wakayama Medical College, Wakayama, Japan. METHODS: Phacoemulsification and aspiration of the crystalline lens followed by implantation of a poly(methyl methacrylate) intraocular lens (IOL) were performed in 1 eye each of 6 albino rabbits; the eye was enucleated 1 or 2 months later. Crystalline lenses were also extracted from the eyes of 2 rabbits. These samples were processed for immunohistochemical detection of the alpha- and beta-subunits of prolyl 4-hydroxylase. RESULTS: A monolayer of LECs was detected on the inner surface of the intact anterior capsule. Antibodies directed against both subunits of prolyl 4-hydroxylase reacted strongly to LECs proliferating on capsules with IOLs, whereas little or no reaction was observed in quiescent LECs or in the regenerated lenticular structure. CONCLUSION: The presence of prolyl 4-hydroxylase in LECs proliferating on the inner surface of the lens capsule suggests that these cells are involved in the production of procollagen and fibrosis during capsular injury and repair. Suppression of prolyl 4-hydroxylase activity may prevent the capsule opacification that results from cataract removal and IOL implantation.

• Vuorela A, Myllyharju J, Nissi R, Pihlajaniemi T, Kivirikko KI
• Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase.
• EMBO J. 1997; 16: 6702-12
• Display abstract

• Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.

• Myllyharju J, Kivirikko KI
• Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.
• EMBO J. 1997; 16: 1173-80
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.

• Annunen P, Helaakoski T, Myllyharju J, Veijola J, Pihlajaniemi T, Kivirikko KI
• Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.
• J Biol Chem. 1997; 272: 17342-8
• Display abstract

• Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha2beta2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered alpha(II) subunit from human sources. The mRNA for the alpha(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (alpha(II))2beta2 tetramer was demonstrated in cultured human WI-38 and HT-1080 cells. The type II tetramer was found to represent about 30% of the total prolyl 4-hydroxylase in these cells and about 5-15% in various chick embryo tissues. The results of coexpression in insect cells argued strongly against the formation of a mixed alpha(I)alpha(II)beta2 tetramer. PDI/beta polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/beta polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source for a simple large scale purification of the recombinant enzyme. The properties of the purified human type II enzyme were very similar to those of the type I enzyme, but the Ki of the former for poly(L-proline) was about 200-1000 times that of the latter. In agreement with this, a minor difference, about 3-6-fold, was found between the two enzymes in the Km values for three peptide substrates. The existence of two forms of prolyl 4-hydroxylase in human cells raises the possibility that mutations in one enzyme form may not be lethal despite the central role of this enzyme in the synthesis of all collagens.

• Veijola J, Pihlajaniemi T, Kivirikko KI
• Co-expression of the alpha subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble alpha-subunit-BiP complexes have no prolyl 4-hydroxylase activity.
• Biochem J. 1996; 315: 613-8
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, their beta subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-beta subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble alpha subunits in solution. We report here that expression of the alpha subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble alpha-subunit-BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the alpha subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the alpha subunit. Nevertheless the soluble alpha-subunit-BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-beta subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the alpha subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation.

• Lamberg A et al.
• Characterization of human type III collagen expressed in a baculovirus system. Production of a protein with a stable triple helix requires coexpression with the two types of recombinant prolyl 4-hydroxylase subunit.
• J Biol Chem. 1996; 271: 11988-95
• Display abstract

• An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless, most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase activity. Pro-alpha 1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the Tm of the triple helices was only about 32-34 degrees C. Coexpression of the pro-alpha1(III) chains with the alpha and beta subunits of human prolyl 4-hydroxylase increased the Tm to about 40 degrees C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-alpha 1(III) chains and alpha1(III) chains were found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced up to about 80 microg of cellular type III collagen (120 microg of procollagen) per 5 X 10(6) cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The 4-hydroxyproline content and Tm of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.

• Koivunen P et al.
• ERp60 does not substitute for protein disulphide isomerase as the beta-subunit of prolyl 4-hydroxylase.
• Biochem J. 1996; 316: 599-605
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha 2 beta 2 tetramers while the Caenorhabditis elegans enzyme is an alpha beta dimer. The beta-subunit is identical to protein disulphide isomerase (PDI), a multifunctional endoplasmic reticulum luminal polypeptide. ERp60 is a PDI isoform that was initially misidentified as a phosphatidylinositol-specific phospholipase C. We report here on the cloning and expression of the human and Drosophila ERp60 polypeptides. The overall amino acid sequence identity and similarity between the processed human ERp60 and PDI polypeptides are 29% and 56% respectively, and those between the Drosophila ERp60 and human PDI polypeptides 29% and 55%. The two ERp60 polypeptides were found to be similar to human PDI within almost all their domains, the only exception being the extreme C-terminal region. Nevertheless, when the human or Drosophila ERp60 was expressed in insect cells together with an alpha-subunit of human prolyl 4-hydroxylase, no tetramer was formed and no prolyl 4-hydroxylase activity was generated in the cells. Additional experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human ERp60 and PDI polypeptides demonstrated that the differences in the C-terminal region are not the only reason for the lack of prolyl 4-hydroxylase tetramer formation by ERp60.

• Mazzorana M, Snellman A, Kivirikko KI, van der Rest M, Pihlajaniemi T
• Involvement of prolyl 4-hydroxylase in the assembly of trimeric minicollagen XII. Study in a baculovirus expression system.
• J Biol Chem. 1996; 271: 29003-8
• Display abstract

• We have shown previously that hydroxylation played a critical role in the trimer assembly and disulfide bonding of the three constituent alpha chains of a minicollagen composed of the extreme C-terminal collagenous (COL1) and noncollagenous (NC1) domains of type XII collagen in HeLa cells (Mazzorana, M., Gruffat, H., Sergeant, A., and van der Rest, M. (1993) J. Biol. Chem. 268, 3029-3032). We have further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric disulfide-bonded type XII minicollagen in an insect cell expression system. For this purpose, type XII minicollagen was produced in insect cells from baculovirus vectors, alone or together with wild-type human prolyl 4-hydroxylase or with the human enzyme mutated in the catalytic site of its alpha or beta subunits or with the individual alpha or beta subunits. When type XII minicollagen was produced alone, negligible amounts of disulfide-bonded trimers were found to be produced by the cells. However, coproduction of the collagen with the two subunits of the wild-type human enzyme dramatically increased the amount of disulfide-bonded trimeric type XII minicollagen molecules. In contrast, coproduction of the collagen with alpha subunits that had a mutation completely inactivating the human enzyme failed to enhance the trimer assembly. These results directly show that an active prolyl 4-hydroxylase is required for the assembly of disulfide-bonded trimers of type XII minicollagen.

• Veijola J, Annunen P, Koivunen P, Page AP, Pihlajaniemi T, Kivirikko KI
• Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their beta subunit.
• Biochem J. 1996; 317: 721-9
• Display abstract

• Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the beta subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/beta polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/beta polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/beta polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/beta and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/beta polypeptide forms an active prolyl 4-hydroxylase alpha 2 beta 2 tetramer with the human alpha subunit and an alpha beta dimer with the C. elegans alpha subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either alpha subunit. Removal of the 32-residue C-terminal extension from the C. elegans alpha subunit totally eliminated alpha beta dimer formation. The C. elegans PDI/beta polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans alpha subunits than did the human PDI/beta polypeptide, being particularly ineffective with the C. elegans alpha subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/beta polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/beta polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase alpha beta dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the K(m) for the hydroxylation of long polypeptide substrates.

• John DC, Bulleid NJ
• Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the alpha-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the beta-subunit.
• Biochem J. 1996; 317: 659-65
• Display abstract

• Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important alpha-subunit and the beta-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an alpha 2 beta 2 tetramer from newly synthesized alpha-subunits associating with an endogenous pool of beta-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled alpha-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018-14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized alpha-subunits were shown to assemble with the beta-subunit, to form insoluble aggregates, or to remain soluble but not associate with the beta-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and alpha-subunits were co-immunoprecipitated with antibodies against the alpha-subunit and BiP, respectively. These results indicate that unassembled alpha-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.

• Saika S et al.
• Immunolocalization of prolyl 4-hydroxylase in fibroblasts cultured from Tenon's capsule of humans.
• Graefes Arch Clin Exp Ophthalmol. 1996; 234: 251-7
• Display abstract

• BACKGROUND: The excessive accumulation of extracellular matrix (ECM) with the repopulation of fibroblasts may lead to an unsuccessful outcome of glaucoma filtering surgery. We examined the immunolocalization of ECM components and prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, in cultured Tenon's capsule fibroblasts (TCFs) of humans to evaluate the production of ECM in the cells. METHODS: We used light microscopy to evaluate the immunolocalization of prolyl 4-hydroxylase and ECM components, collagen types I, III, and IV, cellular fibronectin, and laminin in TCFs. Ultrastructural localization of the enzyme was also evaluated by electron microscopy. RESULTS: Immunoreactivity with monoclonal antibodies against the alpha and beta subunits of the enzyme or with the polyclonal antibody against it was detected in the cytoplasm of the cells in a fine granular pattern, indicating its localization in the endoplasmic reticulum (ER). Immunoreactivity for the enzyme was detected in the cisternae of the ER on electron microscopy. Types I and III collagen reactivities were also observed in the cytoplasm in a fine granular pattern. Type IV collagen reactivity was present diffusely on the cell surface. The distribution of laminin reactivity in the cytoplasm resembled that of types I and III collagen. Cellular fibronectin reactivity was observed in the ECM in a reticular pattern. CONCLUSION: Prolyl 4-hydroxylase was located in the cisternae of the ER. TCFs produced a variety of ECM components in vitro. The results provide insight into the fibrotic process during scar formation at the site of a bleb following filtering surgery.

• Miyaishi O, Kozaki K, Saga S, Sato T, Hashizume Y
• Age-related alteration of proline hydroxylase and collagen-binding heat shock protein (HSP47) expression in human fibroblasts.
• Mech Ageing Dev. 1995; 85: 25-36
• Display abstract

• Changes in the expression of alpha and beta subunits of proline 4-hydroxylase (PH alpha and PH beta) and HSP47, implicated as a molecular chaperone specific for procollagen processing, were examined in human embryonal fibroblasts in relation to in vitro aging. For this purpose a model with treatments causing the decreased hydroxylation of proline residues in procollagens was used. In cells at a low population doubling level (PDL) induction of PH alpha, PH beta, and HSP47 by depletion of ascorbate or addition of alpha-alpha' dipyridyl could be clearly demonstrated by immunoprecipitation and Northern blotting. In contrast, the induction of PH alpha and HSP47 expression was markedly attenuated in high PDL cells, indicating an age-related decrease in response to procollagen retention in the ER caused by hypohydroxylation of proline residues of procollagens.

• Lamberg A, Pihlajaniemi T, Kivirikko KI
• Site-directed mutagenesis of the alpha subunit of human prolyl 4-hydroxylase. Identification of three histidine residues critical for catalytic activity.
• J Biol Chem. 1995; 270: 9926-31
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer in which the alpha subunits contribute to most parts of the two catalytic sites. To study the roles of histidine and cysteine residues in this catalytic activity we converted all 5 histidines that are conserved between species, 4 nonconserved histidines, and 3 conserved cysteines of the human alpha subunit individually to serine and expressed the mutant alpha subunits together with the wild-type beta subunit in insect cells by means of baculovirus vectors. Mutation of any of the 3 conserved histidines, residues 412, 483, and 501, inactivated the enzyme completely or essentially completely, with no effect on tetramer assembly or binding of the tetramer to poly(L-proline). These histidines are likely to provide the three ligands needed for the binding of Fe2+ to a catalytic site. Mutation of either of the other 2 conserved histidines reduced the amount of enzyme tetramer by 20-25% and the activity of the tetramer by 30-60%. Mutation of the nonconserved histidine 324 totally prevented tetramer assembly, whereas mutation of the 3 other nonconserved histidines had no effects. Two of the 3 cysteine to serine mutations, those involving residues 486 and 511, totally prevented tetramer assembly under the present conditions, whereas the third, involving residue 150, had only a minor effect in reducing tetramer assembly and activity. The data do not support previous suggestions that cysteine residues are involved in Fe2+ binding sites. Additional mutagenesis experiments demonstrated that the two glycosylated asparagines have no role in tetramer assembly or catalytic activity.

• Helaakoski T, Annunen P, Vuori K, MacNeil IA, Pihlajaniemi T, Kivirikko KI
• Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.
• Proc Natl Acad Sci U S A. 1995; 92: 4427-31
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.

• Yamada S, Kawasaki H
• [Prolyl hydroxylase]
• Nippon Rinsho. 1995; 53: 383-5

• Tschank G, Sanders J, Baringhaus KH, Dallacker F, Kivirikko KI, Gunzler V
• Structural requirements for the utilization of ascorbate analogues in the prolyl 4-hydroxylase reaction.
• Biochem J. 1994; 300: 75-9
• Display abstract

• The ability of structural analogues of ascorbate to serve as substitutes for this reducing agent in the prolyl 4-hydroxylase reaction was studied. In experiments using the purified enzyme, variations of the compounds' side chain were compatible with co-substrate activity. The presence of very large hydrophobic substituents or a positively charged group caused an increase in the observed Km values. A negative charge and smaller modifications did not change the affinity to the enzyme when compared with L-ascorbate. 6-Bromo-6-deoxy-L-ascorbate had a lower Km than the physiological reductant. Substitution at the -OH group in ring position 3 prevented binding to the enzyme. The same pattern of activity was observed when the full and uncoupled prolyl 4-hydroxylase reactions were studied. The Vmax. values with all compounds were similar. The reaction of microsomal prolyl 4-hydroxylase was supported by D-isoascorbate, O6-tosyl-L-ascorbate and 5-deoxy-L-ascorbate, giving the same dose-response behaviour as L-ascorbate itself. Again, 6-bromo-6-deoxy-L-ascorbate gave a lower Km and a similar Vmax. value. L-Ascorbic acid 6-carboxylate produced substrate inhibition at concentrations above 0.3 mM. The Km and Vmax. values calculated from concentrations up to 0.2 mM were similar to those of L-ascorbate. The enzyme activity observed with 6-amino-6-deoxy-L-ascorbate was very low in the microsomal hydroxylation system. The calculated Vmax. value was lower than that of L-ascorbate, suggesting a restriction of the access of this compound to the enzyme.

• Hopkinson I et al.
• The complete cDNA derived sequence of the rat prolyl 4-hydroxylase alpha subunit.
• Gene. 1994; 149: 391-2
• Display abstract

• A rat cDNA encoding the prolyl 4-hydroxylase alpha subunit (P4H alpha) was isolated and sequenced. The primary aa sequence deduced from the nucleotide sequence reveals a 534-aa protein that shows extensive aa identity with the human (88%) and chick (77%) P4H alpha.

• Helaakoski T et al.
• Structure and expression of the human gene for the alpha subunit of prolyl 4-hydroxylase. The two alternatively spliced types of mRNA correspond to two homologous exons the sequences of which are expressed in a variety of tissues.
• J Biol Chem. 1994; 269: 27847-54
• Display abstract

• Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, plays a central role in collagen synthesis as it catalyzes the formation of 4-hydroxyproline residues by the hydroxylation of proline in X-Pro-Gly sequences. We report here that the human gene for the catalytically important alpha subunit is more than 69 kilobase pairs and consists of 16 exons. The exons that encode solely protein sequences vary from 54 to 240 base pairs (bp), and the introns vary from 750 to more than 16,000 bp. The 133 bp of 5'-untranslated sequences of the mRNA are coded by two exons, and these sequences contain inverted repeats with a potential for stem-loop formation, which may be involved in translational control of the expression of this gene. The 5'-flanking region contains a TATa motif at -29 relative to the major transcription site but no CCAAT motif. The 5'-flanking region and the downstream sequences contain several motifs that may act as binding sites for various transcription factors. Evidence has previously been reported for a mutually exclusive alternative splicing of RNA transcripts of this gene. The present data indicate that the mutually exclusive sequences found in the mRNAs are coded by two consecutive, homologous 71-bp exons 9 and 10. These exons are identical in their first 5 bp and the overall identity between them is 61% at the nucleotide level and 58% at the level of the coded amino acids. Both types of mRNA were found to be expressed in all of the tissues studied, but in some tissues the type coding for exon 9 or 10 sequences was more abundant than the other type.

• Baader E, Tschank G, Baringhaus KH, Burghard H, Gunzler V
• Inhibition of prolyl 4-hydroxylase by oxalyl amino acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues.
• Biochem J. 1994; 300: 525-30
• Display abstract

• The potency of oxalyl amino acid derivatives as inhibitors of prolyl 4-hydroxylase was studied in vitro, in isolated microsomes and in chicken embryonic-tissue culture. These compounds represent structural analogues of 2-oxoglutarate in which the -CH2- moiety at C-3 is replaced by -NH-, with or without further structural modifications. The most efficient inhibitor of purified prolyl 4-hydroxylase was oxalylglycine. Its mode of inhibition was competitive with respect to 2-oxoglutarate. The Ki value varied between 1.9 and 7.8 microM, depending on the variable substrate used. Oxalylalanine inhibited purified enzyme with a Ki of 40 microM. Other oxalyl amino acid derivatives showed little inhibitory activity. In microsomes isolated from embryonic chicken bone, oxalylglycine and oxalylalanine inhibited prolyl hydroxylation with IC50 values of 23 and 120 microM respectively. Dimethyloxalylglycine was not an inhibitor of purified prolyl 4-hydroxylase and only weakly active in the microsomal system, but efficiently suppressed hydroxyproline synthesis in embryonic chicken calvaria and lung. The data suggest that dimethyloxalyl amino acids are converted into active inhibitors in intact cells, most likely in the cytoplasmic compartment.

• John DC, Bulleid NJ
• Prolyl 4-hydroxylase: defective assembly of alpha-subunit mutants indicates that assembled alpha-subunits are intramolecularly disulfide bonded.
• Biochemistry. 1994; 33: 14018-25
• Display abstract

• The vital hydroxylation of peptidyl proline residues in collagens and protein with collagen-like amino acid sequences is catalyzed by the tetrameric enzyme prolyl 4-hydroxylase (P4-H). We have previously detailed [John et al. (1993) EMBO J. 12, 1587-1595] the redox-dependent assembly of the catalytically important alpha-subunit (64 kDa) in a cell-free system containing endogenous beta-subunits (PDI, 60 kDa). To identify the origin of this redox-dependent assembly, we have now shown directly by an electrophoretic mobility shift assay that the assembled wild-type protein possesses at least one intramolecular disulfide bond. We also analyzed five alpha-subunit mutants that have single Cys to Ser mutations in one of the five Cys residues present in the wild-type protein and found that (i) subunits mutated at Cys150 or Cys511 formed intramolecular disulfide bonds, whereas subunits mutated at Cys276, Cys293, or Cys486 did not, (ii) mutation of Cys276, Cys293, or Cys486 led to a large reduction in alpha-beta complex formation, (iii) subunits mutated at Cys276, Cys293, Cys486, or Cys511 were recognized by an antiserum raised against an alpha-subunit C-terminal peptide which failed to recognize the assembled wild-type subunit or the assembled subunit mutated at Cys150, and (iv) the assembled complexes fractionated in a similar position to the purified protein on sucrose gradients whereas the assembly-defective mutants formed higher molecular weight aggregates or complexes with other proteins. On the basis of these results, we propose that P4-H alpha-subunits possess an intramolecular disulfide bond between Cys276 and Cys293 that is essential for alpha-beta complex formation.

• Kasper M, Fuller SD, Schuh D, Muller M
• Immunohistological detection of the beta subunit of prolyl 4-hydroxylase in rat and mini pig lungs with radiation-induced pulmonary fibrosis.
• Virchows Arch. 1994; 425: 513-9
• Display abstract

• Polyclonal and monoclonal antibodies to the beta subunit of prolyl 4-hydroxylase, the protein disulphide isomerase, were used to compare the pulmonary cells in 13 normal and in 20 fibrotic rat and mini-pig lungs made fibrotic by X-ray irradiation, using the ABC immunoperoxidase technique. In normal lungs, prominent staining of Clara cells and type II pneumocytes and weaker reactivity with alveolar macrophages, fibroblasts, endothelial and smooth muscle cells were detectable. In pulmonary disease, in which interstitial fibrosis was the characteristic feature, the immunoreactivity was increased in both the epithelial and interstitial cells. Type I pneumocytes remained negative. In the early stages of disease (3 to 4 weeks after irradiation) when little morphological alteration was seen, capillary endothelial cells had already become immunoreactive. These results underline the complex involvement and interaction of different lung cell populations in the process of pulmonary fibrogenesis.

• Kasper M, Schuh D, Muller M
• Immunohistochemical localization of the beta subunit of prolyl 4-hydroxylase in human alveolar epithelial cells.
• Acta Histochem. 1994; 96: 309-13
• Display abstract

• The beta subunit of prolyl 4-hydroxylase, the protein-disulfide isomerase (PDJ), catalyzes the hydroxylation of proline residues of collagens and proteins with collagen-like structure, a step essential for the folding of the procollagen chains to form triple-helices. In the present study, we report the selective immunohistological localization of PDI in type II alveolar and bronchial epithelial cells. The detection of the hidden antigen with the monoclonal antibody 5B5 is usually not successful in paraffin sections but was possible after microwave pretreatment of tissue sections. In cases of severe lung injury (fibrosing alveolitis) enhanced immunoreactivity was found for this enzyme in epithelial, endothelial as well as interstitial cells and in alveolar macrophages. The results indicate a possible involvement of the pulmonary epithelial cells in the upregulation of collagen production during the process of fibrosis.

• Veijola J, Koivunen P, Annunen P, Pihlajaniemi T, Kivirikko KI
• Cloning, baculovirus expression, and characterization of the alpha subunit of prolyl 4-hydroxylase from the nematode Caenorhabditis elegans. This alpha subunit forms an active alpha beta dimer with the human protein disulfide isomerase/beta subunit.
• J Biol Chem. 1994; 269: 26746-53
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI). We report here on the cloning of the catalytically important alpha subunit from Caenorhabditis elegans. This polypeptide consists of 542 amino acids and signal peptide of 16 additional residues. The C. elegans alpha subunit is 25 amino acids longer than the human alpha subunit, mainly because of a 32-amino-acid C-terminal extension present only in the former. The overall amino acid sequence identity between these two alpha subunits is 45%, a 127-amino acid region close to the C terminus being especially well conserved. When the C. elegans alpha subunit was expressed together with the human PDI/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, but surprisingly this C. elegans/human enzyme appeared to be an alpha beta dimer. The specific activity of this C. elegans/human enzyme was comparable with that of the human enzyme, and most of the other catalytic properties were also highly similar. Nevertheless, the C. elegans/human enzyme was not inhibited by poly(L-proline). The data indicate that the multifunctional PDI/beta subunit can form an active prolyl 4-hydroxylase with alpha subunits having marked differences in their amino acid sequences.

• Yamada H, Aida T, Taguchi K, Asano G
• [Expression of type III and IV procollagen, prolyl 4-hydroxylase mRNAs in fibrotic human liver]
• Nippon Rinsho. 1993; 51: 423-7
• Display abstract

• In situ localization of type III, IV procollagen and prolyl 4-hydroxylase mRNAs were examined to clarify the collagen production in fibrotic human liver. These mRNAs were localized in hepatocytes and mesenchymal cells in the areas of liver fibrosis. In alcoholic liver disease, the expression was promoted, and positive cells were diffusely observed in nodules. These results suggest that hepatocytes along with mesenchymal cells produce collagens, and correlate with pericellular fibrosis and sinusoidal capillarization in alcoholic liver disease.

• John DC, Grant ME, Bulleid NJ
• Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.
• EMBO J. 1993; 12: 1587-95
• Display abstract

• Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.

• Suda K et al.
• [Immunohistochemical localization of prolyl hydroxylase in the pancreatic tissue]
• Nippon Shokakibyo Gakkai Zasshi. 1993; 90: 1721-1721

• Guzman NA, Ascari WQ, Cutroneo KR, Desnick RJ
• Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.
• J Cell Biochem. 1992; 48: 172-89
• Display abstract

• Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.

• Cunliffe CJ, Franklin TJ, Hales NJ, Hill GB
• Novel inhibitors of prolyl 4-hydroxylase. 3. Inhibition by the substrate analogue N-oxaloglycine and its derivatives.
• J Med Chem. 1992; 35: 2652-8
• Display abstract

• N-Oxaloglycine (3) is an alpha-ketoglutarate (1) analogue that is a competitive inhibitor of prolyl 4-hydroxylase (EC 1.14.11.2). A study of the structure-activity relationships of some other oxalo derivatives shows that substitution on the glycine moiety modulates activity stereoselectively and that if the omega-carboxylate is homologated or replaced by either acylsulfonamides or anilide, then activity is sharply reduced. This sensitivity to these changes is contrasted with the relative insensitivity of another putative alpha-ketoglutarate analogue, pyridine-2,5-dicarboxylic acid (2), and the implication is discussed that compounds of both series are unlikely to bind to prolyl hydroxylase in the same way even though both inhibit the enzyme competitively.

• Nagai Y, Kato M, Toda G
• Selectivity of serum immunoreactive prolyl 4-hydroxylase as a marker for hepatic necrosis.
• J Gastroenterol Hepatol. 1992; 7: 253-6
• Display abstract

• To investigate the selectivity of serum immunoreactive prolyl 4-hydroxylase (S-IRPH) as a marker for hepatic necrosis, sera were taken from patients with hepatic inflammation and hepatic necrosis. The concentration of immunoreactive prolyl 4-hydroxylase in the sera was determined by radio-immunoassay, and S-IRPH and alanine aminotransferase (ALT) data were plotted. Patients with hepatic necrosis showed mainly prolyl 4-hydroxylase elevation while patients with inflammation had both prolyl 4-hydroxylase and alanine aminotransferase elevations. The addition of serum immunoreactive prolyl 4-hydroxylase to current serum markers would be useful in investigating the pathophysiology of hepatocellular damage, particularly in differentiating between hepatic necrosis and inflammation.

• Dowell RI, Hadley EM
• Novel inhibitors of prolyl 4-hydroxylase.
• J Med Chem. 1992; 35: 800-4
• Display abstract

• A series of 5-acyl sulfonamides derived from pyridine-2,5-dicarboxylic acid (15) has been prepared and several members of this series have been shown to be more potent, in vitro, as inhibitors of prolyl 4-hydroxylase than 15. Several chain-extended pyridinedicarboxylic acids have also been prepared and shown to be potent inhibitors of prolyl 4-hydroxylase. The structure-activity in both these series is discussed. The results indicate that the 5-carboxylic acid binding site, in the enzyme, can accept a carboxylic acid or an acyl sulfonamide equally well. This indicates a much greater degree of freedom in this distal carboxylic acid binding site than is predicted by the current theoretical model of the active site.

• Tucker H, Thomas DF
• Novel inhibitors of prolyl 4-hydroxylase. 2. 5-Amide substituted pyridine-2-carboxylic acids.
• J Med Chem. 1992; 35: 804-7
• Display abstract

• A series of 5-[(arylcarbonyl)amino]- and 5-(arylcarbamoyl)pyridine-2-carboxylic acids has been prepared and tested for activity as inhibitors of the enzyme prolyl 4-hydroxylase (EC 1.14.11.2). All the analogues prepared were inhibitors of the enzyme in vitro, the best compounds being equipotent with the known inhibitor pyridine-2,5-dicarboxylic acid (9). Like 9 these amidic analogues were not active in a cultured embryonic chick tendon cell model, considered to be a predictor of in vivo activity. The activity of the amides is not consistent with the model described for the mode of action of 9 with the enzyme and aspects of this are discussed.

• Chida Y, Ishizaki M, Nakazawa M, Kao WW
• Expression and methylation of the beta-subunit gene of prolyl 4-hydroxylase: in erythrocytes, tendon and cornea of chick embryos.
• Connect Tissue Res. 1992; 28: 191-204
• Display abstract

• It has recently been demonstrated that the beta-subunit of prolyl 4-hydroxylase (E.C. 1.14.11.2) is the same gene product as protein disulfide isomerase (PDI) and cellular thyroid hormone binding protein (THP). Therefore, it is very likely that the beta-subunit of the prolyl 4-hydroxylase gene serves as a house keeping gene in most cell types. In the present study, we examined the distribution of the chicken beta-subunit of prolyl 4-hydroxylase/protein disulfide isomerase (CPH beta/PDI) in erythrocytes, corneas and tendons of 13-, 17-, and 19-day-old chick embryos by immunohistochemistry using antibodies against CPH beta/PDI. Our data indicate that erythrocytes do not express the CPH beta/PDI gene whereas tendon cells express CPH beta/PDI at all developmental stages examined. The basal cells of corneal epithelium express CPH beta/PDI, but the superficial cell layers of stratified corneas of 19-day-old chick embryos do not. The expression of the CPH beta/PDI gene is also confirmed by in situ hybridization with cDNA encoding CPH beta/PDI. The results indicate that the expression of CPH beta/PDI in cornea is probably developmentally regulated. It has been suggested that methylation of genomic DNA is one of many possible regulatory mechanisms for gene expression. In order to examine whether methylation of genomic DNA may play any role in the expression of the beta-subunit gene, genomic DNA was isolated from corneas, tendons, and erythrocytes of individual 13-, 17-, and 19-day-old chick embryos. DNA samples were digested with Sma I and Eco RI, or Pst I and Sma I and followed by either Msp I, Hpa II, or Hha I and were then subjected to Southern hybridization with 32P-labeled genomic DNA fragments of CPH beta/PDI. Our results indicate that the CPH beta/PDI gene is methylated at the Hha I site in the 4th exon in erythrocytes whereas the same sites in tendon and cornea are hypomethylated. Examination of 5'-end flanking sequences of exon 1 of the CPH beta/PDI gene with the methylation sensitive endonucleases, Hha I and Hpa II did not reveal any difference in erythrocyte, cornea and tendon cells. Thus, our results indicated that DNA methylation may not play an important role in the expression of CPH beta/PDI.

• Myllyla R, Gunzler V, Kivirikko KI, Kaska DD
• Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases.
• Biochem J. 1992; 286: 923-7
• Display abstract

• A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.

• Vuori K, Pihlajaniemi T, Myllyla R, Kivirikko KI
• Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells.
• EMBO J. 1992; 11: 4213-7
• Display abstract

• Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

• Vuori K, Pihlajaniemi T, Marttila M, Kivirikko KI
• Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.
• Proc Natl Acad Sci U S A. 1992; 89: 7467-70
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.

• Myllyla R, Pihlajaniemi T, Pajunen L, Turpeenniemi-Hujanen T, Kivirikko KI
• Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase.
• J Biol Chem. 1991; 266: 2805-10
• Display abstract

• Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.

• Bassuk JA, Berg RA
• Correlation of the steady-state RNA levels among the alpha-subunit of prolyl 4-hydroxylase and the alpha 1 and alpha 2 chains of type I collagen during growth of chicken embryo tendon fibroblasts.
• Biochem Biophys Res Commun. 1991; 174: 169-75
• Display abstract

• The relative steady-state levels of RNAs encoding type I collagen and prolyl 4-hydroxylase were examined in exponentially growing primary cultures of chicken embryo tendon fibroblasts. The RNA levels of the alpha 1 and alpha 2 chains of type I collagen were maximal when the fibroblasts reached the confluent state. The RNA levels of the alpha-subunit of prolyl 4-hydroxylase were also maximal at confluency and rose and fell with the RNA levels of the two collagen chains. The RNA levels of the beta-subunit of prolyl 4-hydroxylase did not correlate with the changes observed for the alpha-subunit or for either chain of type I collagen. The RNA levels of the beta-subunit were slightly higher than the RNA levels of the alpha-subunit. These results support our hypothesis that the synthesis of the alpha-subunit and thus the association of newly synthesized alpha-subunits with pre-existing beta-subunits is the rate-limiting factor in determining prolyl 4-hydroxylase activity in cultured cells.

• Yeowell HN, Murad S, Pinnell SR
• Hydralazine differentially increases mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase whereas it decreases pro alpha 1(I) collagen mRNAs in human skin fibroblasts.
• Arch Biochem Biophys. 1991; 289: 399-404
• Display abstract

• We have used specific oligonucleotide probes to measure the effect of hydralazine on mRNA levels of the alpha and beta subunits of prolyl 4-hydroxylase (PH), a key post-translational modifying enzyme in collagen biosynthesis. Hydralazine exerts a paradoxical effect on collagen biosynthesis in cultured fibroblasts. Cells exposed to hydralazine synthesize substantially reduced amounts of collagen, which is severely deficient in hydroxyproline. Surprisingly, however, the level of prolyl hydroxylase activity assayed in extracts of treated cells is markedly increased, suggesting overproduction of the enzyme. Hybridization analysis indicated that in untreated cells the concentration of the alpha PH subunit mRNA was about 20-25% of the beta PH subunit mRNA concentration. Hydralazine treatment increased the mRNAs for both alpha and beta subunits of PH by three- to fourfold. A differential induction of these mRNAs was observed, however. The alpha subunit mRNA was maximally increased within 24 h, whereas the beta subunit mRNA was increased more slowly, reaching a maximum at 72 h. In contrast, the 5.8 and 4.8-kb mRNAs for pro alpha 1(I) collagen were virtually eliminated by 72 h. This study demonstrates that the increased prolyl hydroxylase activity is a direct result of hydralazine-mediated increases in steady state mRNA content for the alpha and beta subunits of this enzyme. Moreover, the earlier induction of alpha PH mRNA may provide the first evidence at the mRNA level that regulation of PH activity occurs mainly through regulation of the alpha subunit of PH. In addition, the decrease in collagen synthesis by hydralazine appears to result directly from suppression of both species of mRNA for pro alpha 1(I) collagen.

• Tschank G et al.
• Inhibition of prolyl hydroxylation and procollagen processing in chick-embryo calvaria by a derivative of pyridine-2,4-dicarboxylate. Characterization of the diethyl ester as a proinhibitor.
• Biochem J. 1991; 275: 469-76
• Display abstract

• The biochemical and morphological consequences of procollagen prolyl 4-hydroxylase inhibition by pyridine-2,4-dicarboxylic acid (2,4-PDCA) and its diethyl ester (diethyl-2,4-PDC) were studied in chick-embryo calvaria, which predominantly synthesize type I collagen. Half-maximal inhibition of tissue hydroxyproline formation required 650 microM-2,4-PDCA, whereas the Ki with respect to chicken prolyl 4-hydroxylase in vitro was 2 microM. In contrast, half-maximal inhibition was caused by 10 microM-diethyl-2,4-PDC in the intact calvaria, although chicken prolyl 4-hydroxylase in vitro was not inhibited even at 1 mM. The collagenous material produced in the presence of diethyl-2,4-PDC showed an altered 'melting' profile and a lowering of the transition temperature by 10 degrees C, indicating misalignment and thermal instability of its triple-helical structure. Amount and electrophoretic mobility of procollagen type I chains were increased in a dose-dependent manner. The amounts of partially processed species and alpha-chains were decreased, without change in mobility. This marked effect on procollagen-collagen conversion in the intact calvaria suggests that the underhydroxylated collagenous material generated in the presence of diethyl-2,4-PDC is resistant to or acts as endogenous secondary inhibitor of type I procollagen N-proteinase. Electron microscopy of treated calvaria cells showed dilated rough endoplasmic reticulum and numerous phagolysosomes, indicating intracellular retention and lysosomal degradation of the newly synthesized underhydroxylated collagenous material. In summary, these results identify 2,4-PDCA and diethyl-2,4-PDC as the first prolyl 4-hydroxylase-directed inhibitor/proinhibitor pair that affects intra- and extra-cellular events during collagen formation.

• Atreya PL, Ananthanarayanan VS
• Interaction of prolyl 4-hydroxylase with synthetic peptide substrates. A conformational model for collagen proline hydroxylation.
• J Biol Chem. 1991; 266: 2852-8
• Display abstract

• With the aim of understanding the structural basis for the substrate specificity of collagen prolyl 4-hydroxylase, we have studied the conformational features of synthetic oligopeptide substrates and their interaction with the enzyme purified from chicken embryo. Circular dichroism and infrared spectral data, taken in conjunction with relevant crystal structure data, indicated an equilibrium mixture of the polyproline-II (PP-II) helix, the beta-turn, and the random coil conformations in aqueous and trifluoroethanol solutions of the "collagen-related" peptides: t-Boc-Pro-Pro-Gly-Pro-OH, t-Boc-Pro-Pro-Gly-Pro-NHCH3, t-Pro-Pro-Gly-Pro-Pro-OH, t-Boc-Pro-Pro-Ala-Pro-OH, and t-Boc-Pro-Pro-Gln-Pro-OCH3, where t-Boc is tert-butoxycarbonyl. In another set of peptides related to elastin, t-Boc-Val-Pro-Gly-Val-OH and t-Boc-Gly-Val-Pro-Gly-Val-OH, the data indicated the beta-structure, rather than the PP-II helix, was in equilibrium with the beta-turn. Kinetic parameters for the enzymatic hydroxylation of the peptides showed that as a group, the first (proline-rich) set of peptides has higher Km values and lower Vmax and Kcat/Km values than the valine-rich peptides. Data on the inhibition of hydroxylation of the standard assay substrate (Pro-Pro-Gly)10 by the oligopeptides pointed to common binding sites for the peptides. Hydroxyproline-containing peptides had no effect on the hydroxylation of the standard substrate, showing the absence of product inhibition. Based on these and earlier data, we propose that in collagen and related peptides, a supersecondary structure consisting of the PP-II helix followed by the beta-turn is the minimal structural requirement for proline hydroxylation. The PP-II structure would aid effective interaction at the substrate binding subsites, while the beta-turn would be essential at the catalytic site of the enzyme. In elastin and related peptides, the beta-strand structure may be interchangeable with the PP-II structure. This conformational model for proline hydroxylation resolves the discrepancies in earlier proposals on the substrate specificity of prolyl 4-hydroxylase. It is also consistent with the available information on the active site geometry of the enzyme.

• Bassuk JA, Berg RA
• A novel 53-kDa polypeptide from chicken embryo.
• J Biol Chem. 1991; 266: 23732-8
• Display abstract

• We have isolated from chicken embryos a novel 53-kDa protein possessing properties which are similar, but not identical to the 55-kDa PDI polypeptide from chicken embryos. The novel 53-kDa polypeptide copurifies with PDI, but is separated by ion-exchange chromatography. The novel 53-kDa polypeptide cross-reacts strongly with antibodies specific for bovine PDI and cross-reacts to varying degrees with six different preparations of antibodies specific for chicken PDI which is identical to the beta-subunit of chicken prolyl 4-hydroxylase. Anti-bovine PDI immunoglobulins selected by the purified 53-kDa polypeptide react with bovine PDI but not with the beta-subunit of prolyl 4-hydroxylase, suggesting that the 53-kDa polypeptide shares epitopes with bovine PDI but not with the chicken prolyl 4-hydroxylase beta-subunit. Amino acid compositional analysis of the purified polypeptide yielded unique data when compared to PDI and other PDI-like polypeptides. Edman degradation from the N terminus of the 53-kDa polypeptide yields a sequence very different from the N terminus of PDI. This sequence is unique when compared to all entries in available databases. A 20-residue sequence of an internal cyanogen bromide fragment of the 53-kDa polypeptide gives a nearly identical match with human beta-endorphin. The 53-kDa polypeptide is capable of cleaving the disulphides of insulin under conditions where PDI is active. The periodic acid-Schiff assay failed to detect bound carbohydrate. These observations support evidence for a family of PDI-like proteins in chicken embryo and suggest that PDI activity is not confined to only one protein.

• Pihlajaniemi T, Myllyla R, Kivirikko KI
• Prolyl 4-hydroxylase and its role in collagen synthesis.
• J Hepatol. 1991; 13: 27-27
• Display abstract

• Excessive accumulation of collagen in the extracellular matrix has a crucial role in fibrosis. Thus pharmacological inhibition of collagen deposition is likely to be beneficial for patients suffering from fibrotic disorders such as liver cirrhosis. Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of proline residues in -X-Pro-Gly- sequences. The reaction products, 4-hydroxyproline residues, serve to stabilize the collagen triple helices under physiological conditions. Conversely, collagen chains that contain no 4-hydroxyproline cannot fold into triple helical molecules that are stable at body temperature. The prolyl 4-hydroxylase reaction therefore seems to be a particularly suitable target for the pharmological regulation of excessive collagen formation. The reaction catalyzed by prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. The active enzyme is an alpha 2 beta 2 tetramer that consists of two types of inactive monomer and has two catalytic sites. Some parts of the catalytic sites may be built up cooperatively of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit contains the carboxyl-terminal tetrapeptide sequence -Lys-Asp-Glu-Leu which is essential for the retention of a polypeptide within the lumen of the endoplasmic reticulum. Since the alpha subunit lacks the carboxyl-terminal retention signal, one function of the beta subunit in the prolyl 4-hydroxylase tetramer may be to retain the enzyme within the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

• Hanauske-Abel HM
• Prolyl 4-hydroxylase, a target enzyme for drug development. Design of suppressive agents and the in vitro effects of inhibitors and proinhibitors.
• J Hepatol. 1991; 13: 81516-81516
• Display abstract

• The hydrophilic compound pyridine 2,4-dicarboxylate (2,4-PDCA), designed as a mechanism-based competitive inhibitor of prolyl 4-hydroxylase, is efficiently excluded by the cytoplasmic membrane, but permeates the endoplasmic membrane via a 2,4-PDCA-selective translocator to reach its target enzyme in the intracisternal space. A lipophilic 2,4-PDCA-based proinhibitor, inactive with purified prolyl 4-hydroxylase, shows a cell system-dependent suppression of hydroxyprolyl formation, displaying a half-maximally inhibitory concentration very similar to the Ki of the parent compound. Apparently, cell-specific intracellular metabolic processing of the proinhibitor regenerates the active agent, 2,4-PDCA. The in vitro findings summarized here suggest that the 2,4-PDCA-mediated inhibition of prolyl 4-hydroxylase has a marked disruptive effect on the biosynthesis and deposition of collagen. This effect qualifies 2,4-PDCA and its derivatives as experimental fibrosuppressive compounds. However, to avoid catastrophic consequences in vivo, it is desirable to target the active agent to only the tissue that is compromised by excessive matrix formation. This requirement can be realized by the deliberate selection of an appropriate, 2,4-PDCA-based proinhibitor and by the deliberate selection of the route of proinhibitor administration.

• Pajunen L et al.
• Regional assignment of the human gene coding for a multifunctional polypeptide (P4HB) acting as the beta-subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase to 17q25.
• Cytogenet Cell Genet. 1991; 56: 165-8
• Display abstract

• Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens and related proteins by hydroxylating proline residues in peptide linkages. The beta-subunit of prolyl 4-hydroxylase (P4HB) is a highly unusual multifunctional polypeptide that is identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and is highly similar to a glycosylation site-binding polypeptide of oligosaccharyl transferase. We report here the regional assignment of the gene for this multifunctional polypeptide. In situ hybridization mapped the gene to 17q25. Southern blot analyses of restricted DNA from a chromosome-mediated gene transfer transfectant panel suggested that the P4HB gene is located distal to the gene for thymidine kinase, either between the genes for thymidine kinase and galactokinase or on the telomeric side of both these genes.

• de Jong L, van der Kraan I, de Waal A
• The kinetics of the hydroxylation of procollagen by prolyl 4-hydroxylase. Proposal for a processive mechanism of binding of the dimeric hydroxylating enzyme in relation to the high kcat/Km ratio and a conformational requirement for hydroxylation of -X-Pro-Gly- sequences.
• Biochim Biophys Acta. 1991; 1079: 103-11
• Display abstract

• Prolyl 4-hydroxylase modifies only approx. 5% of the hydroxylatable prolyl residues in procollagen at a relatively high rate, after which the rate of further hydroxylation rapidly decreases. This suggests that the probability to exist in a defined hydroxylation-committed conformation differs between the numerous -X-Pro-Gly- sequences in the substrate. The enzyme reaction is characterized by the unusually high kcat/Km ratio of 3 x 10(9) M-1 s-1. To explain these kinetic features, an extremely high second-order rate constant for the association of enzyme and the subset of rapidly hydroxylated prolyl residues has to be assumed. A two-step mechanism is proposed in which diffusional constraints on the rate of association of prolyl 4-hydroxylase with hydroxylatable prolyl residues can be overcome. Upon encountering a random coil pro-alpha chain, the dimeric enzyme is first 'aspecifically' bound, followed by rapid transfers between different segments of the flexible peptide substrate via fast transitions between 'aspecific' single and double bound intermediate states. The rate of the second step, the productive (specific) binding of hydroxylation-committed -X-Pro-Gly- sequence to the active site, can be enhanced significantly by such an, in essence, 'one-dimensional' search. This processive mechanisms of binding does not necessarily imply many hydroxylation reactions during one encounter between enzyme and a peptide with several substrate sites as suggested previously in a slightly different model (De Waal, A. and De Jong, L. (1988) Biochemistry 27, 150-155).

• Karvonen K et al.
• Specific inactivation of prolyl 4-hydroxylase and inhibition of collagen synthesis by oxaproline-containing peptides in cultured human skin fibroblasts.
• J Biol Chem. 1990; 265: 8415-9
• Display abstract

• The crucial role of collagen in fibrotic disorders has prompted attempts to develop drugs that inhibit collagen accumulation. Peptides containing the unphysiological amino acid 5-oxaproline (Opr) have recently been found to act as specific syncatalytic inactivators of pure prolyl 4-hydroxylase, the enzyme that catalyzes the formation of 4-hydroxyproline in collagens. The present study indicates that oxaproline-containing peptides benzyloxycarbonyl-Phe-Opr-Gly-benzyl ester (I) and benzyloxycarbonyl-Phe-Opr-Gly-ethyl ester (II) inactivate prolyl 4-hydroxylase in cultured human skin fibroblasts, peptide I being about twice as potent as peptide II. Inactivation by 50% was observed after culturing with about 20-40 microM concentrations of peptide I for 48 h. The inactivation appears to be specific, as no changes were found in the activities of two other intracellular enzymes of collagen synthesis, lysyl hydroxylase and galactosylhydroxylysyl glucosyltransferase. Synthesis of 4-hydroxyproline by the cells was markedly decreased, and 4-hydroxyproline-deficient procollagen accumulated intracellularly, whereas no changes were found in the incorporation of [14C]leucine into protein after culturing of the cells with a 30 microM concentration of peptide I for 48 h. No changes were seen in the viability of the cells or the release of lactate dehydrogenase from them into the culture medium. No significant changes were found in the steady-state levels of the mRNAs for the pro-alpha 1 chains of type I and type III procollagens or for the alpha and beta subunits of prolyl 4-hyroxylase or fibronectin after culturing with 75 microM peptide I for 48 h. The data indicate that inactivation of cellular prolyl 4-hydroxylase has marked effects on cellular 4-hydroxyproline formation and collagen secretion but no effects on the steady-state levels of mRNAs for type I and III procollagens or the two types of subunit of prolyl 4-hydroxylase.

• Kaule G, Gunzler V
• Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1-14C]succinate: application to prolyl 4-hydroxylase.
• Anal Biochem. 1990; 184: 291-7
• Display abstract

• A method for the assay of 2-oxoglutarate decarboxylating enzymes based on determination of the reaction product [1-14C]succinate after precipitation of remaining 2-oxo[5-14C]glutarate with 2,4-dinitrophenyl hydrazine is reported. It is particularly useful for the study of the 2-oxoglutarate-coupled dioxygenase prolyl 4-hydroxylase (EC 1.14.11.2); it is superior to previously described assay methods of this enzyme with respect to simplicity of the procedure, speed, cost, and radiochemical safety. The results are highly reproducible, the standard deviation of repeated measurements being about 2% of the mean. The commercially available 2-oxo[5-14C]glutarate used in this study contained approximately 3% of radioactivity coeluting with succinate in HPLC and 1.5% of an unidentified radioactive compound as impurities, which contributed to the background.

• Kivirikko KI et al.
• Molecular biology of prolyl 4-hydroxylase.
• Ann N Y Acad Sci. 1990; 580: 132-42

• Myllyla R, Kaska DD, Kivirikko KI
• The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity.
• Biochem J. 1989; 263: 609-11
• Display abstract

• Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens. The beta subunit is known to be identical with the enzyme protein disulphide-isomerase and to possess disulphide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer. We here report that lysyl hydroxylase, a homodimer, and algal prolyl 4-hydroxylase, a monomer, do not contain detectable protein disulphide-isomerase activity. Since the hydroxylase reaction mechanisms are similar, the data suggest that the protein disulphide-isomerase activity of the vertebrate prolyl 4-hydroxylase beta subunit is unlikely to be involved in the catalytic mechanism of the hydroxylation reaction.

• Franklin TJ, Hitchen M
• Inhibition of collagen hydroxylation by 2,7,8-trihydroxyanthraquinone in embryonic-chick tendon cells.
• Biochem J. 1989; 261: 127-30
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. 2,7,8-Trihydroxyanthraquinone (THA) is an effective inhibitor of prolyl 4-hydroxylase by virtue of its ability to compete with the co-substrate 2-oxoglutarate (Ki = 40.3 microM). Using a simple and reproducible assay for collagen hydroxylation, we show that THA inhibits the hydroxylation of collagen in embryonic-chick tendon cells in short-term culture, with an IC50 value (concn. giving 50% inhibition) of 32 microM. In comparison, the ethyl ester of 3,4-dihydroxybenzoic acid has an IC50 value of 0.1 mM against collagen hydroxylation by chick tendon cells, whereas its Ki versus isolated prolyl 4-hydroxylase is 49 microM.

• Helaakoski T, Vuori K, Myllyla R, Kivirikko KI, Pihlajaniemi T
• Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts.
• Proc Natl Acad Sci U S A. 1989; 86: 4392-6
• Display abstract

• Prolyl 4-hydroxylase [procollagen-proline, 2-oxyglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2], an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the alpha-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the beta-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Glu-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha-subunit does not have this C-terminal sequence, and thus one function of the beta-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Interestingly, three of the cDNA clones for the alpha-subunit contained a 64-nucleotide sequence homologous but not identical to the corresponding 64-nucleotide sequence found in four other cDNA clones. Nuclease S1 mapping experiments demonstrated that this difference was due to the existence of two types of mRNA present in approximately equal amounts. Southern blot analyses of human genomic DNA with a cDNA probe for the alpha-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

• Bassuk JA et al.
• Prolyl 4-hydroxylase: molecular cloning and the primary structure of the alpha subunit from chicken embryo.
• Proc Natl Acad Sci U S A. 1989; 86: 7382-6
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2). The beta subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the alpha subunit. One cDNA clone (alpha 1) was isolated from a chicken embryo cDNA expression library in lambda gt11 by screening with anti-alpha-subunit polyclonal immunoglobulins. This alpha 1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken alpha-subunit polypeptide verified that alpha 1 cDNA encoded the alpha subunit. Polymerase chain reactions were used to extend the sequence of alpha 1 cDNA toward the 5' end of alpha-subunit mRNA. The mature alpha subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the alpha subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the alpha subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.

• Kivirikko KI, Myllyla R, Pihlajaniemi T
• Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit.
• FASEB J. 1989; 3: 1609-17
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in X-Pro-Gly sequences. The reaction requires Fe2+, 2-oxoglutarate, O2, and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. Ascorbate is not consumed during most catalytic cycles, but the enzyme also catalyzes decarboxylation of 2-oxoglutarate without subsequent hydroxylation, and ascorbate is required as a specific alternative oxygen acceptor in such uncoupled reaction cycles. A number of compounds inhibit prolyl 4-hydroxylase competitively with respect to some of its cosubstrates or the peptide substrate, and recently many suicide inactivators have also been described. Such inhibitors and inactivators are of considerable interest, because the prolyl 4-hydroxylase reaction would seem a particularly suitable target for chemical regulation of the excessive collagen formation found in patients with various fibrotic diseases. The active prolyl 4-hydroxylase is an alpha 2 beta 2 tetramer, consisting of two different types of inactive monomer and probably containing two catalytic sites per tetramer. The large catalytic site may be cooperatively built up of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit has been found to be identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and shows partial homology with a phosphoinositide-specific phospholipase C, thioredoxins, and the estrogen-binding domain of the estrogen receptor. The COOH-terminus of this beta subunit has the amino acid sequence Lys-Asp-Glu-Leu, which was recently suggested to be necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha subunit does not have this COOH-terminal sequence, and thus one function of the beta subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle.

• Pajunen L et al.
• Assignment of the gene coding for the alpha-subunit of prolyl 4-hydroxylase to human chromosome region 10q21.3-23.1.
• Am J Hum Genet. 1989; 45: 829-34
• Display abstract

• Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages and plays a crucial role in the synthesis of these proteins. The gene for the beta-subunit of prolyl 4-hydroxylase has recently been mapped to the long arm of human chromosome 17, at band 17q25. We report here chromosomal localization of the gene for the catalytically and regulatorily important alpha-subunit of human prolyl 4-hydroxylase. Analysis of 24 rodent x human cell hybrids by Southern blotting with cDNA probes for the human alpha-subunit indicated complete cosegregation of the gene for the alpha-subunit with human chromosome 10. A cell hybrid containing only part of chromosome 10 mapped the gene to 10q11----qter. In situ hybridization mapped the gene to 10q21.3-23.1. The gene for the alpha-subunit is thus not physically linked to that for the beta-subunit of the enzyme.

• Aida T, Asano G
• Synthesis of prolyl 4-hydroxylase by cultured chicken corneal cells.
• Nippon Ika Daigaku Zasshi. 1989; 56: 361-4
• Display abstract

• Prolyl hydroxylase (EC 1.14.11.2) synthesis was examined in the cultured corneas of 17-day-old chick embryos which were cultured in F-12 medium supplemented with fetal calf serum. The amount of prolyl hydroxylase in cells was measured by immunoelectrophoresis using antibodies directed against this enzyme. The results indicated that the amounts of prolyl hydroxylase depended on cell density. The cells were harvested at the early-log phase and plated at high cell density to mimic the culture condition of cells at the stationary phase (crowding). The amount of 35S-prolyl hydroxylase indicated a two-fold increase in the rate of prolyl hydroxylase synthesis after crowding. By use of 32P-labeled cDNA for the beta-subunit of prolyl hydroxylase a proportionate increase of the mRNA was noted in accordance with the increase in the rate of prolyl hydroxylase synthesis, suggesting that this enzyme synthesis is regulated at the transcriptional level.

• Gunzler V, Brocks D, Henke S, Myllyla R, Geiger R, Kivirikko KI
• Syncatalytic inactivation of prolyl 4-hydroxylase by synthetic peptides containing the unphysiologic amino acid 5-oxaproline.
• J Biol Chem. 1988; 263: 19498-504
• Display abstract

• Peptides containing the unphysiological amino acid 5-oxaproline (Opr) in the sequence R1-Xaa-Opr-Gly-OR2 were found to inactivate prolyl 4-hydroxylase from chick and human origins. Of the substances investigated, compounds with aromatic substituents R1 and R2 were particularly effective when compared with those with an aliphatic group or without a C-terminal blocking group. Both affinity of the individual peptides for the enzyme and partition ratio contributed to the differences in efficiency. Benzylcarbonyl-Phe-Opr-Gly-benzyl ester was the most effective substance tested, its concentration giving 50% inactivation in 1 h being 0.8 microM. Inactivation was only observed in the presence of 2-oxoglutarate and Fe2+. The Opr peptides enhanced the decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase, the Vmax values obtained with the individual peptides being positively correlated with their inactivating efficiency. Inactivation was prevented by high concentrations of peptide substrate and ascorbate. Lineweaver-Burk kinetics experiments suggested noncompetitive inhibition with respect to peptide substrate and ascorbate. Lysyl hydroxylase was not affected by Opr peptides in concentrations of up to 1.5 mM in either the presence or absence of prolyl 4-hydroxylase. The results suggest that the oxaproline compounds are specific syncatalytic inactivators of prolyl 4-hydroxylase.

• de Waal A, de Jong L
• Processive action of the two peptide binding sites of prolyl 4-hydroxylase in the hydroxylation of procollagen.
• Biochemistry. 1988; 27: 150-5
• Display abstract

• The number of peptide binding sites of prolyl 4-hydroxylase was manipulated with the peptide photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5, and the effect on hydroxylation of the relatively short peptide substrate (Pro-Pro-Gly)5 and of the long natural substrate procollagen was studied. With (Pro-Pro-Gly)5 as a substrate, a linear relation was found between enzyme activity and the amount of covalently bound photoaffinity label, approximately 50% inactivation being reached at 1 mol of label/mol of enzyme. No difference in Km value for (Pro-Pro-Gly)5 was detected between unlabeled and partially labeled enzyme preparations. These results indicate that enzyme molecules with only one free active site hydroxylated the synthetic substrate (Pro-Pro-Gly)5 with the same Km and at half the rate of native enzyme. In contrast, with procollagen as a substrate a 5-10-fold increase in Km was found with the fraction of enzyme containing only one free active site, as compared to the Km for procollagen with nonlabeled enzyme. This finding is explained by an enzyme-kinetic model based on a processive action of the two peptide substrate binding sites of prolyl 4-hydroxylase, preventing dissociation of the enzyme-substrate complex between successive hydroxylations of a long peptide with multiple substrate sites. Such a mechanism leads to a low Km for a long peptide by overcoming the diffusional constraints on the rate of association between the enzyme and the individual substrate sites.

• Nakazawa M, Aida T, Everson WV, Gonda MA, Hughes SH, Kao WW
• Structure of the gene encoding the beta-subunit of chicken prolyl 4-hydroxylase.
• Gene. 1988; 71: 451-60
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) converts peptidyl proline to peptidyl hydroxyproline in procollagen polypeptides during collagen biosynthesis. The active enzyme is a tetramer which is composed of two pairs of non-identical subunits in a molecular form of alpha 2 beta 2. In addition to the tetrameric prolyl 4-hydroxylase (alpha 2 beta 2), the free beta-subunit is also found inside cells. Recently it was shown that the beta-subunit of prolyl 4-hydroxylase is identical to the protein disulfide isomerase and cellular thyroid hormone-binding protein. We previously isolated and characterized cDNAs of the beta-subunit of chicken prolyl 4-hydroxylase. The cDNA of beta-subunit was used to screen a chicken genomic DNA library constructed with the lambda EMBL-3 vector. Two clones, lambda gCPH beta-22 and beta-50, were isolated and characterized by restriction enzyme analysis, heteroduplex analysis, and nucleotide sequencing. The results showed that the 2.5-kb mRNA of the beta-subunit is divided into eleven exons and that the gene is 9.0 kb long. The gene contains consensus sequence for TATA at -24 bp and four CAAT at -57, -157, -194 and -223 bp in the 5' end flanking sequence of the transcription start point. In addition, there are three GC boxes upstream from the TATA box and four GC boxes in the first intron. This is similar to the structure of the alpha 1(I) collagen coding gene (COL1A1). These elements may interact with nuclear factors and play important roles in expression regulation of the beta-subunit gene as has been described in COL1A1.

• Gunzler V, Hanauske-Abel HM, Myllyla R, Kaska DD, Hanauske A, Kivirikko KI
• Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines.
• Biochem J. 1988; 251: 365-72
• Display abstract

• The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.

• Kaska DD, Myllyla R, Gunzler V, Gibor A, Kivirikko KI
• Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the alpha subunit of the vertebrate enzyme.
• Biochem J. 1988; 256: 257-63
• Display abstract

• Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.

• Tasanen K, Parkkonen T, Chow LT, Kivirikko KI, Pihlajaniemi T
• Characterization of the human gene for a polypeptide that acts both as the beta subunit of prolyl 4-hydroxylase and as protein disulfide isomerase.
• J Biol Chem. 1988; 263: 16218-24
• Display abstract

• A single polypeptide acts as the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase and may also function as a cellular thyroid hormone binding protein. We report here that the human gene for this polypeptide is about 18 kilobase pairs and consists of 11 exons. The two thioredoxin-like regions are coded by exons 1-2 and 8-9, respectively. The codons for the two presumed active sites of protein disulfide isomerase, each a Cys-Gly-His-Cys sequence, are located 12 base pairs from the beginning of exons 2 and 9. The last 3 amino acids coded by exons 1 and 8 and the first 9 amino acids coded by exons 2 and 9, including a broken codon for Tyr, are identical in the respective exon-intron junctions. These regions are also highly homologous to the active sites of bacterial thioredoxins. The data suggest that evolution of this gene has involved exon shuffling and duplication of a two-exon unit, in which the internal exon-intron junctions have been entirely conserved. The region between exons 1-2 and 8-9 appears to contain other duplications. The 5' flanking sequences contain a TATA box, six CCAAT boxes, and other elements which may be involved in regulation of the cellular amounts of this polypeptide.

• Kao WW et al.
• Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase.
• Connect Tissue Res. 1988; 18: 157-74
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.

• Pihlajaniemi T et al.
• Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.
• EMBO J. 1987; 6: 643-9
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.

• Marumo K, Waite JH
• Prolyl 4-hydroxylase in the foot of the marine mussel Mytilus edulis L.: purification and characterization.
• J Exp Zool. 1987; 244: 365-74
• Display abstract

• The mussel foot secretes a variety of unusual hydroxyproline-containing collagenous and noncollagenous proteins. Prolyl 4-hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. Mr of the intact enzyme was 230,000 (alpha 2 beta 2) composed of two subunits with Mr of 60,000 (alpha) and 57,000 (beta) as estimated by HPLC gel filtration and SDS-PAGE. The enzyme utilized (Pro-Pro-Gly)10 as a substrate with an apparent Km value of 0.17 mM. Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8-8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20 degrees C. No detectable hydroxylation occurred with poly-L-proline or the unhydroxylated decapeptide analog (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) of the polyphenolic protein. Kinetic studies, however, revealed that the mussel prolyl 4-hydroxylase was competitively inhibited by poly-L-proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzyme-substrate i.e. (Pro-Pro-Gly)10 complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.

• de Waal A, Hartog AF, de Jong L
• Photoaffinity labelling of the 2-oxoglutarate binding site of prolyl 4-hydroxylase with 5-azidopyridine-2-carboxylic acid.
• Biochim Biophys Acta. 1987; 912: 151-5
• Display abstract

• The synthesis of the photoaffinity label 5-azidopyridine-2-carboxylic acid is described. The 2-oxoglutarate analogue photoaffinity label is a competitive inhibitor with respect to 2-oxoglutarate with a Ki value of 9 X 10(-3) M. Upon ultraviolet irradiation, 5-azidopyridine-2-carboxylic acid inactivated prolyl 4-hydroxylase irreversibly by up to 50%. The extent of inactivation depended on the 5-azidopyridine-2-carboxylic acid concentration and the irradiation time. Inactivation was prevented in the presence of an excess of 2-oxoglutarate. It is concluded that the 5-azidopyridine-2-carboxylic acid became covalently bound to the alpha subunit of prolyl 4-hydroxylase, as the alpha subunit of the photoaffinity labelled enzyme had a decreased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.

• Koivu J, Myllyla R, Helaakoski T, Pihlajaniemi T, Tasanen K, Kivirikko KI
• A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase.
• J Biol Chem. 1987; 262: 6447-9
• Display abstract

• A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.

• Kaska DD, Gunzler V, Kivirikko KI, Myllyla R
• Characterization of a low-relative-molecular-mass prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii.
• Biochem J. 1987; 241: 483-90
• Display abstract

• Prolyl 4-hydroxylase was partially purified and characterized from the unicellular green alga, Chlamydomonas reinhardii. This enzyme differed from all the animal and plant prolyl 4-hydroxylases studied so far in that its Mr was only about 40,000 by gel filtration, being thus less than one-sixth of those determined for the vertebrate and higher-plant enzymes. The algal enzyme did not hydroxylate to any significant extent chick-embryo protocollagen or triple-helical (Pro-Pro-Gly)10, whereas a low hydroxylation rate was found with denatured (Pro-Pro-Gly)10. Poly(L-proline), which is an effective inhibitor of the vertebrate enzymes but acts as a substrate for some higher-plant enzymes, was a good substrate. In the absence of poly(L-proline) the enzyme catalysed an uncoupled decarboxylation of 2-oxoglutarate. Studies of the Km values for the co-substrates and cofactors and the specificity of the 2-oxoglutarate requirement, as well as inhibition studies with selected 2-oxoglutarate analogues, suggested that the catalytic site of the algal enzyme is similar to, but not identical with, those of the vertebrate enzymes. The existence of distinct similarities was further demonstrated by an inhibition of the algal enzyme activity with a monoclonal antibody to the beta-subunit of human prolyl 4-hydroxylase. The amount of prolyl 4-hydroxylase activity in the algal cells was not altered by signals which recognize the presence or absence of the cell wall, as determined in studies on experimental cell-wall regeneration and wall-less mutants.

• Gunzler V, Hanauske-Abel HM, Myllyla R, Mohr J, Kivirikko KI
• Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism.
• Biochem J. 1987; 242: 163-9
• Display abstract

• From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit.

• Cunliffe CJ, Franklin TJ
• Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.
• Biochem J. 1986; 239: 311-5
• Display abstract

• Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. We have found that anthraquinones with at least two hydroxy groups ortho to each other are potent inhibitors of this enzyme. Kinetic studies revealed that 2,7,8-trihydroxyanthraquinone (THA) competitively inhibited the co-substrate, 2-oxoglutarate, but was non-competitive with regard to ascorbate and was tentatively considered to be uncompetitive with regard to protocollagen. The inhibition by THA was greatly enhanced in the absence of added Fe2+ and was partially reversed by the addition of concentrations of Fe2+ in excess of the optimum for the enzymic reaction. Binding studies indicated that THA is an effective chelating agent for Fe2+. Several non-quinoidal compounds bearing the catechol moiety also inhibited the enzyme. The results suggest that THA inhibited prolyl 4-hydroxylase by binding to the enzyme at the site for 2-oxoglutarate possibly involving the Fe2+ atom, rather than by complexing with Fe2+ in free solution. The inhibition of prolyl 4-hydroxylase by THA exhibited strong positive co-operativity and may involve three distinct but non-independent binding sites.

• Koivu J, Myllyla R
• Protein disulfide-isomerase retains procollagen prolyl 4-hydroxylase structure in its native conformation.
• Biochemistry. 1986; 25: 5982-6
• Display abstract

• Protein disulfide-isomerase was isolated as a homogeneous protein from 15-day-old chick embryos. The enzyme has a molecular weight of 56,000 in SDS-polyacrylamide gel electrophoresis. Its Km value for randomly cross-linked ribonuclease, a protein used as a substrate for the enzyme, was 0.3 microM, and the Km value for DTT was 1.0 microM. Its optimum pH was 7.5 and its optimum temperature, 33 degrees C. The maximal velocity of pure protein disulfide-isomerase from chick embryos under optimal conditions was about 29,000 units/g. Protein disulfide-isomerase was able to activate purified prolyl 4-hydroxylase 2- to 3-fold, the activation being higher for enzyme stored for a longer time. This activation is probably due to the repairing of disulfide exchanges occurring in the prolyl 4-hydroxylase structure during purification and storage. Prolyl 4-hydroxylase activity was very stable in microsomes, however, and protein disulfide-isomerase was unable to increase the microsomal prolyl 4-hydroxylase activity, suggesting that prolyl 4-hydroxylase retains its native conformation in microsomes. Protein disulfide-isomerase was able to reactivate prolyl 4-hydroxylase inactivated by mild H2O2 treatment. The activity obtained after this treatment and protein disulfide-isomerase incubation corresponded to the amount of prolyl 4-hydroxylase tetramer found after H2O2 treatment. The data suggest that protein disulfide-isomerase is able to activate only the tetramer part of the enzyme preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

• Majamaa K, Gunzler V, Hanauske-Abel HM, Myllyla R, Kivirikko KI
• Partial identity of the 2-oxoglutarate and ascorbate binding sites of prolyl 4-hydroxylase.
• J Biol Chem. 1986; 261: 7819-23
• Display abstract

• Various hydroxybenzenes, hydroxybenzoic acids, and related compounds resemble structurally both 2-oxoglutarate and ascorbate, two reactants needed in the reaction of prolyl 4-hydroxylase. These substances were found to inhibit prolyl 4-hydroxylase competitively with respect to both cosubstrates. Ortho-dihydroxy derivatives, which are capable of chelating the enzyme-bound iron, were the most effective inhibitors, with Ki values of about 5 microM. In contrast, pyridine 2-carboxylates, which have previously been reported to inhibit the enzyme competitively with respect to 2-oxoglutarate, were found to inhibit it uncompetitively with respect to ascorbate. In a separate set of experiments the side chain of the ascorbate molecule was shown to make no significant contribution to the binding of the reductant to the enzyme, as D(-)-isoascorbate and 5,6-O-isopropylidene ascorbate gave essentially the same Vmax and Km values as ascorbate. On the other hand, structural modifications of the ring atoms that abolished the chelating capacity destroyed both the cosubstrate and inhibitory activity, as in L-galactono gamma-lactone. The ascorbate binding site therefore appears to consist of two cis-positioned coordination sites of the enzyme-bound iron and is thus partially identical to the binding site of 2-oxoglutarate. This mode of interaction suggests that ascorbate reduces the enzyme-bound iron through an "inner-sphere" mechanism. The inhibitors studied appear to react at different phases of the catalytic cycle, determined by the oxidation state of the enzyme-bound iron atom.

• Bai Y, Muragaki Y, Obata K, Iwata K, Ooshima A
• Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase.
• J Biochem (Tokyo). 1986; 99: 1563-70
• Display abstract

• Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4-hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme.

• Yoshida S et al.
• A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody.
• Clin Chim Acta. 1986; 160: 37-46
• Display abstract

• Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody (Fab') to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng (0.4 fmol) of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng (0.04 fmol) per tube, and linearity was obtained between 0.01 to 30 ng (0.04-125 fmol) per tube. The radioimmunoassay used a 125I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. The enzyme immunoassay gave reproducible quantitation and evidenced a higher enzyme concentration in the serum of patients with liver disorders. Protein immunoblotting showed that the serum immunoreactive prolyl 4-hydroxylase trapped in the sandwich immunoassay was mainly the beta-subunit.

• Majamaa K et al.
• Differences between collagen hydroxylases and 2-oxoglutarate dehydrogenase in their inhibition by structural analogues of 2-oxoglutarate.
• Biochem J. 1985; 229: 127-33
• Display abstract

• Inhibition of lysyl hydroxylase and prolyl 3-hydroxylase was studied with 23 selected aromatic and aliphatic structural analogues of 2-oxoglutarate and the results were compared with those previously reported for prolyl 4-hydroxylase. All the compounds inhibited the hydroxylases competitively with respect to 2-oxoglutarate and noncompetitively with respect to Fe2+ and the peptide substrate. The inhibition patterns for the three collagen hydroxylases were basically similar, but certain differences in detail emerged. One systematic difference was that lysyl hydroxylase had a higher Ki for almost all the compounds than had the two prolyl hydroxylases. Another interesting difference was that pyridine-2,4-dicarboxylate was the most potent inhibitor of lysyl hydroxylase and prolyl 3-hydroxylase, with Ki values of 50 microM and 3 microM respectively, whereas pyridine-2,5-dicarboxylate was the most potent inhibitor of prolyl 4-hydroxylase. These and other data suggest that the three collagen hydroxylases have similar but not identical 2-oxoglutarate-binding sites. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate and their corresponding benzene derivatives were also found to inhibit 2-oxoglutarate dehydrogenase, but with this enzyme, unlike the collagen hydroxylases, no distinct difference in the Ki values was found between the corresponding pyridine and benzene derivatives. This demonstrates the importance of the metal ion for the binding of various compounds at the 2-oxoglutarate-binding site of the collagen hydroxylases. 2-Oxoadipate was shown to replace 2-oxoglutarate in the lysyl hydroxylase and 2-oxoglutarate dehydrogenase reactions, as has previously been reported for prolyl 4-hydroxylase, whereas no other 2-oxo acid tested had any co-substrate activity. The 2-oxoglutarate-binding site of these enzymes is thus flexible to a certain degree, as it can accommodate molecules of different shapes and volumes. On the basis of the present data pyridine-2,5-dicarboxylate seems to be a quite specific inhibitor of prolyl 4-hydroxylase, the Ki for 2-oxoglutarate dehydrogenase being about 4000-fold higher.

• Kivirikko KI, Majamaa K
• Synthesis of collagen: chemical regulation of post-translational events.
• Ciba Found Symp. 1985; 114: 34-64
• Display abstract

• Collagen biosynthesis involves many unique post-translational events. Inhibition of some of these will lead either to decreased formation of the extracellular collagen fibres or to an accumulation of fibres with altered functional properties. The events that would seem most suitable targets for chemical regulation are triple helix formation, the cleavage of propeptides from the procollagen molecules and cross-link formation. Attempts have recently been made to develop inhibitors of prolyl 4-hydroxylase in particular, as inhibition of this enzyme will prevent triple helix formation and thus lead to a non-functional protein. Prolyl 4-hydroxylase is inhibited competitively with respect to ferrous ion by several bivalent cations, especially zinc, with respect to 2-oxoglutarate by pyridine 2,5-dicarboxylate, pyridine 2,4-dicarboxylate, 3,4-dihydroxybenzoate and many related compounds, with respect to oxygen by superoxide dismutase-active copper chelates and with respect to the peptide substrate by a number of peptides. Triple helix formation can also be inhibited by administering certain proline analogues such as cis-4-hydroxyproline and L-azetidine-2-carboxylic acid, which are incorporated into proteins in place of proline. Only preliminary data are available on the possibilities for using any of these substances to inhibit collagen accumulation in fibrotic processes.

• de Waal A, de Jong L, Hartog AF, Kemp A
• Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5.
• Biochemistry. 1985; 24: 6493-9
• Display abstract

• The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.

• Hoyhtya M, Myllyla R, Piuva J, Kivirikko KI, Tryggvason K
• Monoclonal antibodies to human prolyl 4-hydroxylase.
• Eur J Biochem. 1984; 141: 472-82
• Display abstract

• Monoclonal antibodies against human prolyl 4-hydroxylase (EC 1.14.11.2), an intracellular enzyme of collagen biosynthesis, were produced by fusing spleen cells from BALB/c mice hyperimmunized with human prolyl 4-hydroxylase and mouse myeloma cells (P3/NS 1/1-AG 4-1). Hybridomas from 14 different primary microtiter-plate well cultures produced antibodies to human prolyl 4-hydroxylase; six of them with the highest antibody titer were cloned and antibodies produced by one clone from each of the six lines were further characterized. All of the six cloned hybrids produced antibodies of the IgG class as detected by immunodiffusion. The enzyme antigen used in the present study was a tetramer composed of two pairs of different subunit proteins, alpha and beta. Only one clone which produced antibodies to the alpha subunit was obtained, the other five antibodies being directed against the beta subunit. All the antibodies reacted with the tetramer form of the enzyme. Species cross-reactivity of the antibodies was tested using cultured human, mouse and chick fibroblasts and purified prolyl 4-hydroxylase from chick and mouse sources. None of the antibodies cross-reacted with chick or mouse fibroblasts, as determined by immunofluorescence, whereas one antibody reacted with purified chick and mouse prolyl 4-hydroxylase when examined by the western blotting technique. This antibody caused a strong inhibition of human prolyl 4-hydroxylase activity, but the other five antibodies had negligible inhibitory effect on the activity of the enzyme.

• Majamaa K, Hanauske-Abel HM, Gunzler V, Kivirikko KI
• The 2-oxoglutarate binding site of prolyl 4-hydroxylase. Identification of distinct subsites and evidence for 2-oxoglutarate decarboxylation in a ligand reaction at the enzyme-bound ferrous ion.
• Eur J Biochem. 1984; 138: 239-45
• Display abstract

• The structure and function of the 2-oxoglutarate binding site of prolyl 4-hydroxylase was studied by assaying the inhibitory potential of 24 selected aliphatic or aromatic compounds. All except one of them inhibited the enzyme competitively with respect to 2-oxoglutarate and noncompetitively with respect to Fe2+, the Ki values ranging from 0.8 microM to over 15 mM. The Ki values for the two most effective inhibitors, pyridine 2,5-dicarboxylate and 2,4-dicarboxylate, were about 0.8 microM and 2 microM, these compounds being the most potent inhibitors of prolyl 4-hydroxylase with respect to 2-oxoglutarate known so far. Only one of the compounds tested, 2-oxoadipinate, was able to support hydroxylation by replacing 2-oxoglutarate as a cosubstrate. The data suggest that the 2-oxoglutarate binding site can be divided into three distinct subsites. Subsite I is probably a positively charged side chain of the enzyme that ionically binds the C5 carboxyl group of the 2-oxoglutarate, subsite II consists of two cis-positioned equatorial coordination sites of the enzyme-bound ferrous ion and is chelated by the C1-C2 moiety, while subsite III involves a hydrophobic binding site in the C3-C4 region of the cosubstrate. The sp3 rehybridization of C2 within the chelating moiety of the cosubstrate appears to be a crucial event during decarboxylation that proceeds in the form of a ligand reaction inside the Fe2+ coordination sphere.

• Turpeenniemi-Hujanen T, Myllyla R
• Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro.
• Biochim Biophys Acta. 1984; 800: 59-65
• Display abstract

• Concomitant hydroxylation of proline and lysine residues in protocollagen was studied using purified enzymes. The data suggest that prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) and lysyl hydroxylase (peptidyllysine, 2-oxoglutarate; oxygen 5-oxidoreductase, EC 1.14.11.4) are competing for the protocollagen substrate, this competition resulting in an inhibition of the lysyl hydroxylase but not of the prolyl 4-hydroxylase reaction. When the same protocollagen was used for these hydroxylases, the affinity of prolyl 4-hydroxylase to the protocollagen substrate was about 2-fold higher than that of lysyl hydroxylase. Hydroxylation of lysine residues in protocollagen had no effect on the affinity of prolyl 4-hydroxylase, whereas hydroxylation of proline residues decreased the affinity of lysyl hydroxylase to one-half of the value determined before the hydroxylation. When enzyme preparations containing different ratios of lysyl hydroxylase activity to prolyl 4-hydroxylase activity were used to hydroxylase protocollagen substrate, it was found that in the case of a low ratio the hydroxylation of lysine residues seemed to proceed only after a short lag period. Accordingly, it seems probable that most proline residues are hydroxylated to 4-hydroxyproline residues before hydroxylation of lysine residues if the prolyl 4-hydroxylase and lysyl hydroxylase are present as free enzymes competing for the same protocollagen substrate.

• Myllyla R, Majamaa K, Gunzler V, Hanauske-Abel HM, Kivirikko KI
• Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase.
• J Biol Chem. 1984; 259: 5403-5
• Display abstract

• The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles.

• De Jong L, Kemp A
• Stoicheiometry and kinetics of the prolyl 4-hydroxylase partial reaction.
• Biochim Biophys Acta. 1984; 787: 105-11
• Display abstract

• In the absence of a peptidylproline substrate, the oxidative decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) is stoicheiometrically coupled to the oxidation of ascorbate. The Km and Kd for O2 in this partial reaction are 1.5 mM, this value being one order of magnitude higher than the Km and Kd for O2 in the complete reaction in the presence of (Pro-Pro-Gly)5, indicating that in this case O2 can become enzyme-bound predominantly after the interaction of the peptide substrate with the enzyme. The Km values for 2-oxoglutarate in the partial and the complete reactions are the same. In the absence of both a peptide substrate and ascorbate 2 mol CO2 per mol enzyme are produced in the first 1-1.5 min, during which the enzyme becomes inactivated and, as shown earlier (De Jong , L., Albracht , S.P.J. and Kemp, A. (1982) Biochim. Biophys. Acta 704, 326-332) enzyme-bound Fe2+ becomes oxidized to Fe3+. The results are consistent with a mechanism in which a Fe2+O complex is the O-transferring intermediate involved in peptidylproline hydroxylation.

• Kainulainen H, Takala T, Myllyla R, Hassinen I, Vihko V
• Increased prolyl 4-hydroxylase activity in the myocardium of endurance-trained mice.
• Experientia. 1983; 39: 1094-5
• Display abstract

• Endurance training over 3, 10 or 20 days increased the activity of prolyl 4-hydroxylase (PH) in the left ventricle of mice. No increase was observed in the weight of the left ventricle, in galactosylhydroxylysyl glucosyltransferase activity or in hydroxyproline concentration. The increase in PH suggests that the synthesis of collagen increases during physiological adaptation of the heart to endurance exercise without changes in the ventricle weight or its total collagen content.

• Nietfeld JJ, de Jong L, Kemp A
• The influence of 2-oxoglutarate on the activity of prolyl 4-hydroxylase.
• Biochim Biophys Acta. 1982; 704: 321-5
• Display abstract

• 1. Treatment of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with 2-oxoglutarate in the absence of added Fe2+ for 10 s causes partial inactivation of the enzyme which is not reversed by subsequent addition of Fe2+. It appears that 2-oxoglutarate prevents loss of enzyme-bound iron and prevents access of added iron to its binding site. 2. For optimal enzyme activity the enzyme should be preincubated for 15 s with Fe2+ (5 microM). 3. Under turnover conditions prolyl 4-hydroxylase does not release iron. 4. The inactivation brought about by pre-incubation with 2-oxoglutarate and O2 in the absence of ascorbate is partly reversed by removal of 2-oxoglutarate. 5. It is proposed that dead-end complex formation with 2-oxoglutarate is responsible for the inactivation of the enzyme by 2-oxoglutarate in the absence of either ascorbate or Fe2+. 6. Optimal enzyme activity is obtained if the reactants are added to the reaction medium in the following order: enzyme, iron and ascorbate in any order, then after at least 15 s 2-oxoglutarate and finally (Pro-Pro-Gly)5 to start the reaction.

• Majamaa K, Oikarinen J
• Labelling of prolyl hydroxylase tetrameric subunits in freshly isolated chick-embryo tendon cells and in certain chick-embryo tissues in vivo.
• Biochem J. 1982; 204: 737-42
• Display abstract

• The labelling of the subunits of prolyl 4-hydroxylase tetramers was studied in freshly isolated chick-embryo tendon cells and in chick-embryo tissues. In the former both the alpha- and beta-subunits of the tetramer were labelled during a 4 h labelling and 2 h chase period, although the radioactivity in the beta-subunit was much lower than in the alpha-subunit. The corresponding subunits of the enzyme from 12-day chick-embryo cartilaginous bone and heart were labelled in 7 h, again the beta-subunit much less than the alpha-subunit, the ratio of radioactivity in the beta-subunit to that in the alpha-subunit (beta/alpha-radioactivity) being 0.20 and 0.32 respectively. The beta/alpha-radioactivity then increased almost linearily with time between 7 and 24 h, by 9.5-fold in the cartilaginous bone and 3-fold in the heart, and beta/alpha-radioactivity values above 1.0 were reached. The free beta-subunit-size protein (the beta'-protein), which is also present in cells, had been labelled quite heavily by 7 h. The beta/alpha-radioactivity at 7h, determined in four tissues with different ratios of prolyl hydroxylase tetramers to total immunoreactive protein (tetramer percentage), was low in tissues with a high tetramer percentage. It is thus proposed that only a minor fraction of the beta'-protein must be processed to the tetrameric beta-subunit and utilized in the synthesis of the prolyl 4-hydroxylase tetramers.

• De Jong L, Kemp A
• Two Fe2+ atoms are present in activated prolyl 4-hydroxylase.
• Biochim Biophys Acta. 1982; 709: 142-5
• Display abstract

• A linear relationship was found between the activity of prolyl 4-hydroxylase (EC 1.14.11.2) and the amount of Fe2+ bound per mol enzyme. At maximal activity (2.1 mumol X min-1 per mg protein) the enzyme contains 2.1 mol Fe2+ specifically bound per mol enzyme tetramer, indicating two Fe2+-binding sites on prolyl 4-hydroxylase. The half-maximal concentration of added Fe2+ for enzyme activity depends on the nature of sulphydryl compounds present in the reaction medium.

• Soskel NT, Kuby SA
• A steady-state kinetic analysis of the prolyl-4-hydroxylase mechanism.
• Connect Tissue Res. 1981; 9: 121-5
• Display abstract

• Published kinetic data by Kivirikko, et al. on the prolyl-4-hydroxylase reaction have been re-evaluated using the overall steady-state velocity equation in the forward and reverse directions for an ordered ter ter kinetic mechanism. Qualitatively, the published data for prolyl-4-hydroxylase appear to fit the predicted patterns for this kinetic mechanism. More kinetic data are needed to confirm these results and to quantitate the kinetic parameters but, tentatively, the order of substrate addition would appear to be alpha-ketoglutarate, oxygen, and peptide; and the order of product release would be hydroxylated peptide (or collagen), carbon dioxide, and succinate.

• Nietfeld JJ, Van der Kraan I, Kemp A
• Dissociation and reassociation of prolyl 4-hydroxylase subunits after cross-linking of monomers.
• Biochim Biophys Acta. 1981; 661: 21-7
• Display abstract

• 1. Incubation of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with H2O2 leads to a decrease of 50% in the specific activity of enzyme tetramers, followed by dissociation into inactive dimers in which the monomers are covalently cross-linked by S-S bridge formation. 2. Incubation of the enzyme with K3Fe(CN)6 leads to a comparable decrease in activity of enzyme tetramers. Addition of urea leads to dissociation into inactive dimers with similarly cross-linked monomers. 3. Removal of the dissociating agent leads to reassociation of cross-linked dimers to tetramers and to about 50% reactivation. The enzyme is further reactivated by preincubation with dithiothreitol. 4. Dissociation of the enzyme with dithiothreitol, urea or LiCl, or at low pH (4.15) produces inactive monomers, which could not be reassociated.

• Nietfeld JJ, Kemp A
• The function of ascorbate with respect to prolyl 4-hydroxylase activity.
• Biochim Biophys Acta. 1981; 657: 159-67
• Display abstract

• 1. Incubation in the presence of 2-oxoglutarate and oxygen inactivates prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2), with a t 1/2 of 80 s at 37 degrees C. This inactivation is not affected by the presence or absence of the prolyl peptide substrate or added Fe(II). 2. This inactivation can be prevented by either ascorbate or dithiothreitol. It can be reversed by dithiothreitol but not by ascorbate. 3. Although the iron-containing form of prolyl 4-hydroxylase requires ascorbate for activity, ascorbate is not stoicheiometrically consumed in the reaction catalysed by the enzyme. Ascorbate cannot be replaced by alloxan, lactate, NADH plus phenazine methosulphate, dithiothreitol or L-cysteine. 4. Ascorbate has a double function with respect to prolyl 4-hydroxylase activity. On the one hand, it is required to initiate the reaction when the enzyme has become oxidized during isolation. On the other hand it is required for the protection against inactivation induced by 2-oxoglutarate and oxygen, presumably by preventing S-S bridge formation. The latter function may be of physiological importance.

• Rokowski R, Cutroneo KR, Guzman NA, Fallon A, Cardinale GJ
• In vitro synthesis of collagen prolyl hydroxylase. The newly synthesized enzyme contains two newly synthesized subunits of the alpha and beta size.
• J Biol Chem. 1981; 256: 1340-5

• Kao W, Chou KL
• CRP, immunologically cross-reacting protein of prolyl hydroxylase. Its role in assembly of active pro-yl hydroxylase and cellular localization in L-929 fibroblasts.
• Arch Biochem Biophys. 1980; 199: 147-57

• Chichester CO, Fuller GC
• Evidence for the presence of an endogenous inhibition of prolyl hydroxylase.
• Res Commun Chem Pathol Pharmacol. 1980; 29: 329-38
• Display abstract

• Prolyl hydroxylase [EC 1.14.11.2] was shown to be inhibited by an ultrafiltrate (less than 30,000 molecular weight) fraction isolated from skin and blood of neonatal and adult rabbits. This fraction also inhibited two other alpha-ketoglutarate requiring mixed function oxidases, lysyl hydroxylase [EC 1.14.11.4] and alpha-butyrobetaine hydroxylase [EC 1.14.11.1] but not the amine oxidase, lysyl oxidase. Purification of the skin ultrafiltrate on Sephadex G-25 demonstrated a peak of prolyl hydroxylase inhibitory activity which chromatographed at a molecular weight corresponding to approximately 3,000. Chromatography of a blood ultrafiltrate separated a similar peak of material which was inhibitory for prolyl hydroxylase.

• Berg RA, Kao WW, Kedersha NL
• The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized alpha-subunits and from preformed cross-reacting protein.
• Biochem J. 1980; 189: 491-9

• Counts DF, Cutroneo KR
• Collagen prolyl hydroxylase activation in dermal cell primary cultures.
• Int J Biochem. 1980; 12: 401-5

• Nietfeld JJ, Kemp A
• Properties of prolyl 4-hydroxylase containing firmly-bound iron.
• Biochim Biophys Acta. 1980; 613: 349-58
• Display abstract

• 1. Prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) was isolated in a form containing iron (0.85-1.1 mol Fe/mol enzyme). 2. The enzyme was pure according to gel electrophoresis and had a high specific activity (1.8-2.6 mumol . mi-1 . mg-1). 3. Experiments with metal chelators showed this iron to be firmly bound and to be required for catalytic activity. 4. According to EPR spectrometry the bound iron is not part of a [2Fe-2S] or a [4Fe-4S] cluster. 5. The enzyme activity is to a large extent independent of added Fe2+. 6. The enzyme activity is completely dependent on ascorbate. 7. In the absence of ascorbate but the presence of substrates the enzyme is irreversibly inactivated. 8. Continuous measurement of enzyme activity was possible by following oxygen uptake.

• Tryggvason K, Majamaa K, Risteli J, Kivirikko KI
• Partial purification and characterization of chick-embryo prolyl 3-hydroxylase.
• Biochem J. 1979; 183: 303-7
• Display abstract

• Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.

• Counts DF, Cardinale GJ, Udenfriend S
• Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate.
• Proc Natl Acad Sci U S A. 1978; 75: 2145-9
• Display abstract

• Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of alpha-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate alpha-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and for study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(L-proline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of alpha-ketoglutarate without itself being hydroxylated. All of these findings prove that alpha-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate.

• Bhatnagar RS, Rapaka RS, Urry DW
• Interaction of polypeptide models of elastin with prolyl hydroxylase.
• FEBS Lett. 1978; 95: 61-4

• Risteli J, Tuderman L, Tryggvason K, Kivirikko KI
• Effect of hepatic injury on prolyl 3-hydroxylase and 4-hydroxylase activities in rat liver and on immunoreactive prolyl 4-hydroxylase concentrations in the liver and serum.
• Biochem J. 1978; 170: 129-35
• Display abstract

• After severe hepatic injury induced by dimethylnitrosamine, approximately a 4-fold increase in hepatic prolyl 4-hydroxylase activity occurred within 4 days, whereas the increases in total immunoreactive prolyl 4-hydroxylase protein and in prolyl 3-hydroxylase activity were only about 1.4-fold. The different magnitudes of the increases in the prolyl 4-hydroxylase and 3-hydroxylase activities were verified after partial purification of the enzymes by gel filtration. The data support previous reports indicating differential increases in the activities of individual enzymes of collagen biosynthesis in hepatic injury. Separation of prolyl 4-hydroxylase tetramers from the monomer-size protein by gel filtration indicated that the increase in enzyme activity was similar to that in enzyme tetramers, and an increase had also occurred in the ratio of enzyme tetramers to total enzyme protein. Thus the specific activity of the tetramers had remained unchanged in liver injury. The administration of dimethylnitrosamine was also accompanied by a marked increase in the immunoreactive prolyl 4-hydroxylase protein concentration in the serum, and a similar effect was also noted after carbon tetrachloride administration, results suggesting that the increases originated in the liver.

• Adams E
• Invertebrate collagens.
• Science. 1978; 202: 591-8
• Display abstract

• The collagens of all major invertebrate phyla have been studied, but characterization has been thorough in only a few classes and in no case in the detail (such as sequence analysis) known for vertebrate collagen. Biochemical data on insect collagen are particularly sparse. Invertebrate and vertebrate collagens are strikingly similar, with some notably unique features in annelids and nematodes. Present data do not support the suggestion that invertebrate collagens resemble vertebrate basement membrane collagen. In invertebrates, as in vertebrates, collagens of specific tissues show differenes that probably reflect individual tissue requirements.

• Chen-Kiang S, Cardinale GJ, Udenfriend S
• Homology between a prolyl hydroxylase subunit and a tissue protein that crossreacts immunologically with the enzyme.
• Proc Natl Acad Sci U S A. 1977; 74: 4420-4
• Display abstract

• A protein, enzymatically inactive but immunologically related to prolyl hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase; EC 1.14.11.2) (cross-reacting protein), has been purified to near homogeneity from skin of newborn rats. The purified protein has a molecular weight of 60,000 on gel filtration and sodium dodecyl sulfate gel electrophoresis, corresponding to that of the smaller of the two dissimilar subunits of the enzyme. The two subunits of prolyl hydroxylase differ markedly from one another in their amino acid compositions, but crossreating protein and the smaller subunit are very similar in composition. On antibody-affinity chromatography both subunits reacted with the antibody developed against the intact enzyme. Neither crossreacting protein nor the 60,000 molecular weight subunit was adsorbed onto concanavalin A, which adsorbed the intact enzyme as well as the larger subunit. It would appear that crossreacting protein is identical to one of the subunits of prolyl hydroxylase or metabolically related to it.

• Cardinale GJ, Udenfriend S
• Prolyl hydroxylase.
• Adv Enzymol Relat Areas Mol Biol. 1974; 41: 245-300

© 2021 EMBL | Privacy Policy