NORMAL GENOMIC

• Zhao C, Nguyen T, Liu L, Sacco RE, Brogden KA, Lehrer RI
• Gallinacin-3, an inducible epithelial beta-defensin in the chicken.
• Infect Immun. 2001; 69: 2684-91
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• Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized, epithelial beta-defensins of the chicken (Gallus gallus) and turkey (Meleagris gallopavo), respectively. In normal chickens, the expression of gallinacin-3 was especially prominent in the tongue, bursa of Fabricius, and trachea. It also occurred in other organs, including the skin, esophagus, air sacs, large intestine, and kidney. Tracheal expression of gallinacin-3 increased significantly after experimental infection of chickens with Haemophilus paragallinarum, whereas its expression in the tongue, esophagus, and bursa of Fabricius was unaffected. The precursor of gallinacin-3 contained a long C-terminal extension not present in the prepropeptide. By comparing the cDNA sequences of gallinacin-3 and GPV-1, we concluded that a 2-nucleotide insertion into the gallinacin-3 gene had induced a frameshift that read through the original stop codon and allowed the chicken propeptide to lengthen. The striking structural resemblance of the precursors of beta-defensins to those of crotamines (highly toxic peptides found in rattlesnake venom) supports their homology, even though defensins are specialized to kill microorganisms and crotamines are specialized to kill much larger prey.

• Hughes AL
• Evolutionary diversification of the mammalian defensins.
• Cell Mol Life Sci. 1999; 56: 94-103
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• Defensins are cysteine-rich cationic peptides that function in antimicrobial defense in both invertebrates and vertebrates. Three main groups of animal defensins are known: insect defensins; mammalian alpha-defensins and vertebrate beta-defensins. It has been difficult to determine whether these molecules are homologous or have independently evolved similar features, but overall the evidence favors a distant relationship. The best evidence of this relationship is structural, particularly from their overall three-dimensional structure and from the spacing of half-cystine residues involved in intra-chain disulfide bonds. Some evidence favors a closer relationship between vertebrate beta-defensins and insect defensins than between the two groups of vertebrate defensins. Examination of nucleotide substitutions between recently duplicated mammalian defensin genes shows that the rate of nonsynonymous (amino-acid-altering) substitution exceeds that of synonymous substitution in the region of the gene encoding the mature defensin. This highly unusual pattern of nucleotide substitution is evidence that natural selection has acted to diversify defensins at the amino acid level. The resulting rapid evolution explains why it is difficult to reconstruct the evolutionary history of these molecules.

• Schnapp D, Reid CJ, Harris A
• Localization of expression of human beta defensin-1 in the pancreas and kidney.
• J Pathol. 1998; 186: 99-103
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• Defensins are antimicrobial peptides which play a key role in innate immunity. High levels of human beta defensin-1 (hBD-1) have previously been detected in the kidney and pancreas, but the cell-specific location of hBD-1 mRNA has not been determined. The expression of hBD-1 mRNA has been examined in fetal and adult pancreas and kidney by mRNA in situ hybridization. In fetal pancreas, hBD-1 expression was detected in the developing acini and in adult pancreas in the acini, but not in the pancreatic ducts. In both fetal and adult kidney, hBD-1 expression was detected in the collecting ducts and in the loops of Henle in adult kidney. Expression of hBD-1 mRNA in the pancreas and kidney from early development and in the acini of the adult pancreas, rather than in the pancreatic ducts, may indicate that in these tissues, hBD-1 fulfils physiological functions in addition to host defence.

• Weinberg A, Krisanaprakornkit S, Dale BA
• Epithelial antimicrobial peptides: review and significance for oral applications.
• Crit Rev Oral Biol Med. 1998; 9: 399-414
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• Epithelial tissues provide the first line of defense between an organism and the environment. Disruption of this barrier leads to bacterial invasion and subsequent inflammation. This is precisely the situation existing in the human oral cavity, where tissues are constantly exposed to a variety of microbial challenges that can lead to bacterially induced periodontal diseases, and to infections of the oral mucosa by bacteria, fungi, and viruses. With the recent discoveries of host-derived peptide antibiotics in mammalian mucosal epithelium, a new line of investigation is emerging to test the hypothesis that one class of these peptides, called "beta-defensins", functions to protect the host against microbial pathogenesis at these critical, confrontational sites. In that light, impairment of beta-defensin activity has recently been implicated in chronic bacterial infections in cystic fibrosis patients. The first direct evidence of expression of defensin peptides in the oral mucosa was the identification of a novel epithelial beta-defensin in mammalian tongue. It was shown to be upregulated in inflammation, suggesting that it participates in host defense. It is theorized that epithelial cell-derived antimicrobial peptides function to keep the natural flora of micro-organisms in a steady state in different niches such as the skin, the intestines, the airway, the endocervix, and the mouth. There is now evidence indicating that normal gingival epithelial cells and tissues express two beta-defensins, hBD-1 and the newly described hBD-2. In addition, a cathelin-class antimicrobial peptide, designated LL-37 and found in human neutrophils, is also expressed in skin and gingiva. It is highly likely that these and/or other epithelial antimicrobial peptides play an important role in determining the outcome of the host-pathogen interaction at the oral mucosal barrier, and that they may have important future applications in antibiotic treatment.

• Valore EV, Park CH, Quayle AJ, Wiles KR, McCray PB Jr, Ganz T
• Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.
• J Clin Invest. 1998; 101: 1633-42
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• Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.

• Huttner KM, Lambeth MR, Burkin HR, Burkin DJ, Broad TE
• Localization and genomic organization of sheep antimicrobial peptide genes.
• Gene. 1998; 206: 85-91
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• Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two beta-defensins and eight cathelicidins. We mapped the two-exon beta-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep-hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.

• Zucht HD et al.
• Human beta-defensin-1: A urinary peptide present in variant molecular forms and its putative functional implication.
• Eur J Med Res. 1998; 3: 315-23
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• Human beta-defensin-1 (hBD-1) was first isolated from blood filtrate by our group. Further studies elucidate the significance of this peptide in the human urogenital tract. The hBD-1 gene is expressed in urogenital epithelial organs such as urinary bladder, ureter, vagina and particularly in distal tubular cells of the kidney. Functional characterization of hBD-1 was carried out with native hBD-1 purified from human body fluids. Several different N-terminally truncated variants derived from the 68-amino acid-containing precursor of hBD-1 occur in blood filtrate and in urine. The generation of these variants can be explained by digestion through a chymotrypsin-like protease. Unlike the alpha-defensins which are structurally related peptide antibiotics, our results indicate that native hBD-1 exhibits minor antimicrobial activity which is not related to the extension of the N-terminus. Only few microorganisms, for example bacilli, are significantly inhibited by hBD-1. Moreover, antibiotic activity is suppressed in solutions containing physiological sodium chloride concentrations. This is in contrast to previous reports assuming a pivotal role of hBD-1 in antimicrobial host defense. In contrast to its weak antimicrobial activity, it is shown that hBD-1 has a strong cytotoxic potential towards mammalian cells like NIH-3T3 fibroblasts. We assume that this property might be important during eradicative processes at epithelia in particular when the synthesis rate of this peptide is upregulated.

• Zhao C, Liaw L, Lee IH, Lehrer RI
• cDNA cloning of Clavanins: antimicrobial peptides of tunicate hemocytes.
• FEBS Lett. 1997; 410: 490-2
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• Clavanins are a family of alpha-helical antimicrobial peptides found in hemocytes of the tunicate, Styela clava. We examined a cDNA library prepared from pharyngeal tissues of S. clava and sequenced 24 clones that encoded prepropeptides of Clavanins A, C, D or E. These sequences indicated that Clavanins are synthesized as 9.2 kDa prepropeptides which contain a 19-residue signal peptide, followed in turn by a highly polar 'pro' region (LEERKSEEEK) with five glutamic acid residues, the 23 residues of the mature Clavanin peptide, the glycine residue needed for its amidation and a 27-residue polar C-terminal extension that is removed in later processing. Although the signal sequence and anionic propiece of Clavanin precursors share features with corresponding regions in precursors of the certain frog peptides, including ranalexin, gaegurins, dermaseptins and deltorphins, their unique multipartite structure suggests that they are not actually homologues of these amphibian peptides.

• Bevins CL, Diamond G
• Molecular biological strategies in the analysis of antibiotic peptide gene families. The use oligonucleotides as hybridization probes.
• Methods Mol Biol. 1997; 78: 151-66

• Yount NY, Wang MS, Yuan J, Banaiee N, Ouellette AJ, Selsted ME
• Rat neutrophil defensins. Precursor structures and expression during neutrophilic myelopoiesis.
• J Immunol. 1995; 155: 4476-84
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• Defensins constitute a family of 3- to 4-kDa antimicrobial peptides that are stored in the cytoplasmic granules of neutrophils, some macrophages, and intestinal Paneth cells. We have assessed defensin gene expression during myeloid differentiation by first characterizing cDNAs for each of the four known rat neutrophil defensins (RatNP 1-4). The cDNA sequences revealed that the peptides are synthesized as 87-94 amino acid precursors, each containing signal, pro-, and mature peptide segments. RatNP-3 and -4 mRNAs, but not those for RatNP-1 and -2 or other myeloid defensins, contained unique polypurine tracts located close to the termination codon in the 3' untranslated region. By using cDNA probes and/or riboprobes, we evaluated defensin transcript levels in a variety of tissues and in the full spectrum of neutrophil precursors. By in situ hybridization, defensin mRNAs were localized to neutrophil precursors in the bone marrow, with the highest mRNA levels occurring in promyelocytes and somewhat lower signals occurring in myeloblasts and myelocytes. Defensin mRNAs were not detectable in bands or mature neutrophils, nor at significant levels in tissues other than bone marrow. The accumulation of defensin protein in bone marrow cells was assessed by immunohistochemical staining with anti-RatNP-1 Ab. RatNP 1-4 mRNAs and protein levels were then correlated for each stage of neutrophilic differentiation to reveal the maturational profile of myeloid defensin gene expression in the rat.

• Ganz T
• Biosynthesis of defensins and other antimicrobial peptides.
• Ciba Found Symp. 1994; 186: 62-71
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• Defensins are small (about 30 amino acid residues) cationic antimicrobial peptides with a conserved framework of six disulphide-linked cysteines. Human defensin HNP-1 and the closely related HNP-3 are amphiphilic dimers that act in part by permeabilizing cell membranes. Defensin mRNAs, abundant in neutrophilic promyelocytes, certain non-human macrophages and Paneth cells, encode 94-100 amino acid prepropeptides. PreproHNP-1 is post-translationally processed to inactive proHNP-1 then to mature HNP-1 stored in granules. Bactenecin Bac-5 and perhaps other related neutrophil peptides are also synthesized as prepropeptides but are stored in granules as inactive propeptides. Their conserved cathelin-like propiece inhibits the cysteine protease, cathepsin L, and is removed only during granule release. Charge neutralization of mature peptide by the propiece is seen in both probactenecins and prodefensins. In contrast the propiece of cecropins is very short and proceropins are microbicidal. The pathways that convert myeloid preprodefensins to defensins are specific to myeloid cells but the signal for targeting to granules also functions in non-myeloid granulated cells. The truncation of the anionic propiece by deletion mutagenesis dramatically reduces defensin synthesis, suggesting that the propiece may assist in peptide stabilization, folding or subcellular transport. Despite some similarities in the mechanism of action of the various families of antimicrobial peptides, their precursors differ greatly, presumably owing to differing functions of the propieces.

• Clark DP, Durell S, Maloy WL, Zasloff M
• Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin.
• J Biol Chem. 1994; 269: 10849-55
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• Antimicrobial peptides comprise a diverse class of molecules used in host defense by plants, insects, and animals. In this study we have isolated a novel antimicrobial peptide from the skin of the bullfrog, Rana catesbeiana. This 20 amino acid peptide, which we have termed Ranalexin, has the amino acid sequence: NH2-Phe-Leu-Gly-Gly-Leu-Ile-Lys-Ile-Val-Pro-Ala-Met-Ile-Cys-Ala-Val-Thr- Lys-Lys - Cys-COOH, and it contains a single intramolecular disulfide bond which forms a heptapeptide ring within the molecule. Structurally, Ranalexin resembles the bacterial antibiotic, polymyxin, which contains a similar heptapeptide ring. We have also cloned the cDNA for Ranalexin from a metamorphic R. catesbeiana tadpole cDNA library. Based on the cDNA sequence, it appears that Ranalexin is initially synthesized as a propeptide with a putative signal sequence and an acidic amino acid-rich region at its amino-terminal end. Interestingly, the putative signal sequence of the Ranalexin cDNA is strikingly similar to the signal sequence of opioid peptide precursors isolated from the skin of the South American frogs Phyllomedusa sauvagei and Phyllomedusa bicolor. Northern blot analysis and in situ hybridization experiments demonstrated that Ranalexin mRNA is first expressed in R. catesbeiana skin at metamorphosis and continues to be expressed into adulthood.