NORMAL GENOMIC

• Oldham NJ, Krieger J, Breer H, Svatos A
• Detection and removal of an artefact fatty acid from the binding site of recombinant Bombyx mori pheromone-binding protein.
• Chem Senses. 2001; 26: 529-31
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• Recombinant Bombyx mori pheromone-binding protein (PBP), purified from an Escherichia coli expression system, has been found to contain (11Z)-octadecenoic acid (cis-vaccenic acid) as an artefact ligand. An efficient delipidation procedure is described to overcome what would appear to be a general problem with recombinant lepidopteran PBPs.

• Zhang Sg, Maida R, Steinbrecht RA
• Immunolocalization of odorant-binding proteins in noctuid moths (insecta, lepidoptera).
• Chem Senses. 2001; 26: 885-96
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• Odorant-binding proteins were studied in the noctuid moths Agrotis segetum, Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis using antisera raised against the pheromone-binding protein (PBP) and general odorant-binding protein 2 (GOBP2) of Antheraea polyphemus (Saturniidae). Proteins immunoreacting with these antisera were only found on the antennae and PBP and GOBP2 could be identified on western blots of males and females of all five species. PBPs were predominantly localized in sensilla trichodea and GOBP2 in sensilla basiconica, in good correlation with the stimulus specificity of the receptor cells in these sensilla. In H. armigera and H. virescens the majority of the s. trichodea immunoreacted with the antiserum against PBP of A. polyphemus; in A. segetum, A. gamma and S. littoralis, on the other hand, a high percentage of s. trichodea remained unlabelled. Probably, the PBP expressed in these sensilla is so different that it does not immunoreact with the antiserum used. Such a protein was found by native PAGE of antennal extracts of A. segetum and S. littoralis. These data correlate with the fact that the two heliothine species use pheromones with the same alkyl chain length as A. polyphemus, while the other three species use pheromones with shorter chains. In H. armigera, H. virescens, A. gamma and S. littoralis female antennae were also immunolabelled and a large number of PBP-expressing s. trichodea was consistently found. In S.littoralis this fits with the electrophysiologically recorded high pheromone sensitivity of female s. trichodea, whereas in females of H. armigera and H. virescens no or only weak responses to pheromone stimulation have been reported. Therefore, PBP expression in a sensillum does not necessarily imply pheromone sensitivity of its receptor cells.

• Maida R, Ziesmann J
• Female Attacus atlas respond to pheromones of Antheraea polyphemus: a comparative electrophysiological and biochemical study.
• Chem Senses. 2001; 26: 17-24
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• Female Attacus atlas respond electrophysiologically to both of the Antheraea polyphemus pheromone components (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-6,11-hexadecadienal. Moreover, they possess a pheromone-binding protein (PBP) and general odorant-binding proteins (GOBPs), as well as a pheromone-degrading sensillar esterase and aldehyde oxidase enzymes. They show no electroantennogram responses to their own gland extract. In contrast, female A. polyphemus do not respond to their own or to A. atlas pheromone. Male A. atlas do not detect any of the A. polyphemus compounds but only the conspecific female gland extracts. Both male A. atlas and female A. polyphemus possess PBP and GOBP but lack the pheromone-degrading esterases of male Antheraea. The results indicate that the two species use quite distinct classes of chemicals as pheromones. In spite of this, the N-terminal amino acid sequences of the PBPs show homology of 68%.

• Campanacci V et al.
• Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay.
• J Biol Chem. 2001; 276: 20078-84
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• Pheromone-binding proteins (PBPs), located in the sensillum lymph of pheromone-responsive antennal hairs, are thought to transport the hydrophobic pheromones to the chemosensory membranes of olfactory neurons. It is currently unclear what role PBPs may play in the recognition and discrimination of species-specific pheromones. We have investigated the binding properties and specificity of PBPs from Mamestra brassicae (MbraPBP1), Antheraea polyphemus (ApolPBP1), Bombyx mori (BmorPBP), and a hexa-mutant of MbraPBP1 (Mbra1-M6), mutated at residues of the internal cavity to mimic that of BmorPBP, using the fluorescence probe 1-aminoanthracene (AMA). AMA binds to MbraPBP1 and ApolPBP1, however, no binding was observed with either BmorPBP or Mbra1-M6. The latter result indicates that relatively limited modifications to the PBP cavity actually interfere with AMA binding, suggesting that AMA binds in the internal cavity. Several pheromones are able to displace AMA from the MbraPBP1- and ApolPBP1-binding sites, without, however, any evidence of specificity for their physiologically relevant pheromones. Moreover, some fatty acids are also able to compete with AMA binding. These findings bring into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal compounds.

• Callahan FE, Vogt RG, Tucker ML, Dickens JC, Mattoo AK
• High level expression of "male specific" pheromone binding proteins (PBPs) in the antennae of female noctuiid moths.
• Insect Biochem Mol Biol. 2000; 30: 507-14
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• Pheromone Binding Proteins (PBPs) are one branch of a multigene family of lepidopteran Odorant Binding Proteins (OBPs) that are known for their relatively high levels of expression in male antennae. However, PBP expression has been observed at low levels in female antennae of the Saturniidae, Bombycidae and Lymantriidae, and at relatively high levels in members of the Noctuiidae. The function of female PBP expression is unclear, as female lepidoptera are consistently noted for their failure to respond physiologically or behaviorally to sex-pheromone. In this study, the sexual dimorphism of PBP expression was examined in the noctuiid moths Helicoverpa zea, Heliothis virescens and Spodoptera frugiperda. A PBP cDNA clone was isolated from female H. zea, PBP-Hzea(f). Northern blot analysis indicated relatively high levels of PBP-Hzea(f) expression in both male and female antennae, though females consistently expressed about 50% that of males. Western blot analysis of male and female PBP expression supported these relative differences. Immunocytochemical analysis indicates discrete expression localized beneath olfactory sensilla of both male and female antennae. These results suggest female noctuiids possess the biochemistry to detect at least components of their sex-pheromone. Alternatively, these results may suggest that PBPs have a more general function in noctuiids, possibly reflecting behavioral and life history differences that distinguish this the Noctuiidae from other Lepidopteran families.

• Plettner E, Lazar J, Prestwich EG, Prestwich GD
• Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar.
• Biochemistry. 2000; 39: 8953-62
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• The gypsy moth, Lymantria dispar, uses (7R, 8S)-cis-2-methyl-7, 8-epoxyoctadecane, (+)-disparlure, as a sex pheromone. The (-) enantiomer of the pheromone is a strong behavioral antagonist. Specialized sensory hairs, sensillae, on the antennae of male moths detect the pheromone. Once the pheromone enters a sensillum, the very abundant pheromone binding protein (PBP) transports the odorant to the sensory neuron. We have expressed the two PBPs found in gypsy moth antennae, PBP1 and PBP2, and we have studied the affinity of these recombinant PBPs for the enantiomers of disparlure. To study pheromone binding under equilibrium conditions, we developed and validated a binding assay. We have addressed the two major problems with hydrophobic ligands in aqueous solution: (1) concentration-dependent adsorption of the ligand on vial surfaces and (2) separation of the protein-bound ligand from the material remaining free in solution. We used this assay to demonstrate for the first time that pheromone binding to PBP is reversible and that the two PBPs from L. dispar differ in their enantiomer binding preference. PBP1 has a higher affinity for the (-) enantiomer, while PBP2 has a higher affinity for the (+) enantiomer. The PBP from the wild silk moth, Antheraea polyphemus (Apol-3) bound the disparlure enantiomers more weakly than either of the L. dispar PBPs, but Apol-3 was also able to discriminate the enantiomers. We have observed extensive aggregation of both L. dispar PBPs and an increase in pheromone binding at high (>2 microM) PBP concentrations. We present a model of disparlure binding to the two PBPs.

• LaForest SM, Prestwich GD, Lofstedt C
• Intraspecific nucleotide variation at the pheromone binding protein locus in the turnip moth, Agrotis segetum.
• Insect Mol Biol. 1999; 8: 481-90
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• Inter- and intraspecific amino acid variability in the pheromone binding proteins (PBPs) of the Lepidoptera is believed to contribute to a molecular mechanism of pheromone blend discrimination. Messenger RNA coding for PBP sequence in Agrotis segetum (Noctuidae) was cloned, and nucleotide and inferred amino acid variation across a 769-bp region of a PBP locus was studied in two populations. A single gene copy was fully sequenced, revealing an intron/exon structure conserved with distant saturniids. While several nucleotide substitutions are predicted to result in amino acid replacement, tests for the presence of natural selection suggest that the observed variation is neutral. A phylogenetic analysis provides evidence that the two populations are in the process of genetic isolation.

• Willett CS, Harrison RG
• Pheromone binding proteins in the European and Asian corn borers: no protein change associated with pheromone differences.
• Insect Biochem Mol Biol. 1999; 29: 277-84
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• Pheromone binding proteins (PBPs) are thought to play a role in the recognition of sex pheromone in male moth antennae. By binding selectively to different components of pheromone blends, these PBPs could play a role in differentiating between structurally related compounds. In this study we have characterized the pheromone binding proteins of two pheromone strains of the European corn borer (Ostrinia nubilalis) and also the closely related Asian corn borer (O. furnacalis). We have been able to detect only one PBP gene, which encodes a mature protein that is identical in amino acid sequence in individuals from different pheromone strains and different species. This result suggests that the PBP is not detecting differences between the two isomeric compounds of the European corn borer pheromone or the difference in double bond position between the pheromone molecules of the European and Asian corn borers.

• Leal WS, Nikonova L, Peng G
• Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori.
• FEBS Lett. 1999; 464: 85-90
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• Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.

• Campanacci V, Longhi S, Nagnan-Le Meillour P, Cambillau C, Tegoni M
• Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1). Functional and structural characterization.
• Eur J Biochem. 1999; 264: 707-16
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• Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) present at high concentrations ( approximately 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. By expression in Escherichia coli, we have obtained large quantities (2-3 mg.L-1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). These quantities are compatible with the requirements of X-ray and NMR studies. The recombinant protein has been characterized by native-polyacrylamide gel electrophoresis, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a structural analogue, Z11-16:TFMK (cis-11-hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend previous findings on PBPs. MbraPBP1 and two PBPs from different species have been found to exist as dimers under nondenaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs rather than the beta-barrel fold found in vertebrates odorant binding proteins. We have tentatively identified the location of the helices and the short beta-strands with respect to the binding site. Currently we have obtained small diffracting crystals of the recombinant MbraPBP1 and determined their space group and molecular content.

• Danty E et al.
• Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein.
• J Neurosci. 1999; 19: 7468-75
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• Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at approximately 2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.

• Maibeche-Coisne M et al.
• Molecular cloning and bacterial expression of a general odorant-binding protein from the cabbage armyworm Mamestra brassicae.
• Eur J Biochem. 1998; 258: 768-74
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• A cDNA clone encoding a general odorant-binding protein (GOBP2) was isolated from antennal RNA of Mamestra brassicae by reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA encoding the GOBP2 was further used for bacterial expression. Most of the recombinant GOBP2 (>90%) was found to be insoluble. Purification under denaturing conditions consisted of solubilisation of inclusion bodies, affinity chromatography, refolding and gel filtration. The refolded rGOBP2 was cross-reactive with a serum raised against the GOBP2 of the Lepidoptera Antheraea polyphemus. The purified refolded rGOBP2 was further characterised by native PAGE, IEF, N-terminal sequencing, and two-dimensional NMR. A functional characterisation of the rGOBP2 was carried out by testing its ability to bind pheromone compounds. The yields of production and purification fulfil the requirements of structural studies.

• Krieger J, von Nickisch-Rosenegk E, Mameli M, Pelosi P, Breer H
• Binding proteins from the antennae of Bombyx mori.
• Insect Biochem Mol Biol. 1996; 26: 297-307
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• From an antennal library of Bombyx mori cDNA clones encoding different binding proteins have been isolated. The deduced amino acid sequences showed only moderate homology to each other but shared several common structural features. Based on a sequence comparison with the antennal binding proteins from different moth species, one of the clones appears to encode a pheromone binding protein, whereas two others represent new members of the two general odorant binding protein families. A fourth clone encodes a protein which is related to antennal binding proteins so far found only in Drosophila melanogaster.

• Ziegelberger G
• The multiple role of the pheromone-binding protein in olfactory transduction.
• Ciba Found Symp. 1996; 200: 267-75
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• Before airborne odorant molecules can stimulate the olfactory receptor cells of animals that live on land, they have to pass through an aqueous solution that contains high concentrations of soluble odorant-binding proteins (OBPs). In insect sensilla the role of these OBPs for signal transduction is becoming multifaceted. Sensillum lymph perfusion experiments in the moth Antheraea polyphemus implied a solubilizer and carrier function of the pheromone-binding protein (PBP) and led to the conclusion that it is the pheromone-PBP complex which activates the postulated receptors. Recent results have shown the presence of two redox states of the PBP and a shift in pheromone binding from the reduced to the oxidized form, depending on the presence of sensory hair material. Thus, PBP oxidation might occur simultaneously with receptor cell activation and might lead to deactivation of the pheromone-PBP complex terminating the pheromone stimulation.

• Varoqui H et al.
• Expression of the vesicular acetylcholine transporter in mammalian cells.
• Prog Brain Res. 1996; 109: 83-95

• Ozaki M, Morisaki K, Idei W, Ozaki K, Tokunaga F
• A putative lipophilic stimulant carrier protein commonly found in the taste and olfactory systems. A unique member of the pheromone-binding protein superfamily.
• Eur J Biochem. 1995; 230: 298-308
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• In chemosensory systems, a variety of lipophilic ligand-binding proteins have been found in saliva or nasal mucus. Lipophilic stimulants reach the receptor membrane, carried by these proteins. An acidic 14-kDa protein purified in the blowfly, Phormia regina, belongs to the insect pheromone-binding protein superfamily, but unlike other lipophilic ligand-binding proteins in insect or vertebrate chemosensory systems, it was distributed in both taste and olfactory organs. A similar protein was also isolated in Drosophila melanogaster. Considering their distributions, cDNA sequences and structural features, we concluded that these proteins belong to a unique subfamily whose members have convergently evolved for a common function required for both senses of taste and olfaction. By an electrophysiological experiment using antiserum, we also suggested that these proteins carry fragrant components of natural foods in taste systems as well as in olfactory systems.

• Du G, Prestwich GD
• Protein structure encodes the ligand binding specificity in pheromone binding proteins.
• Biochemistry. 1995; 34: 8726-32
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• The ligand specificities and binding affinities of three recombinant pheromone binding proteins (PBPs) of two saturniid moths (genus Antheraea) were determined by using a novel binding assay in conjunction with two tritium-labeled constituents of the pheromone blend, [3H]-6E,11Z-hexadecadienyl acetate and [3H]-4E,9Z-tetradecadienyl acetate. The new binding assay, in which nonspecific adsorption to a plastic vessel is suppressed by presaturation of the surface with a 1-alkanol, allows measurement of dissociation constants (KD) for lipophilic ligands for their carrier proteins. The three PBPs showed KD values for [3H]-6E,11Z-16:Ac and [3H]-4E,9Z-14:Ac between 0.6 and 30 microM, as determined by Scatchard analysis. Importantly, two PBPs (Aper-1 and Aper-2) from one species showed opposite binding specificities for these two ligands. Aper-1, like Apol-3, showed 15-fold higher affinity for 6E,11Z-16:Ac than for 4E,9Z-14:Ac, while Aper-2 showed a 3.5-fold preference for binding the shorter chain compound. In addition, for the Apol-3 PBP, displacement of [3H]-6E,11Z-16:Ac binding by other pheromone components or analogs showed a clear trend in relative binding affinity: 6E,11Z-16:Ac > 4E,9Z-14:Ac > 6E,11Z-16:Al approximately 16:Ac > 6E,11Z-16:OH > 4E,9Z-14:OH. These data clearly demonstrate a > 1000-fold range of binding affinities among these very similar structures and unambiguously demonstrate the specificity of the PBP-pheromone interaction. Moreover, this assay offers the potential for determining ligand specificities for odorant binding proteins and other proteins in the vertebrate lipocalin superfamily.

• Ziegelberger G
• Redox-shift of the pheromone-binding protein in the silkmoth Antheraea polyphemus.
• Eur J Biochem. 1995; 232: 706-11
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• In pheromone-sensitive hairs of the male silkmoth Antheraea polyphemus, two electrophoretically distinct pheromone-binding proteins (PBPs) are present. They indicate no amino acid sequence diversity according to peptide mapping, but differ in their redox state, as shown by free-sulfhydryl-group-specific cleavage at cysteine residues with 2-nitro-5-thiocyanobenzoic acid. In kinetic studies, the pheromone was initially bound mainly by the reduced PBP but later by the oxidized PBP, where all six cysteine residues form disulfide bonds. This redox shift was observed only in the homogenate of isolated olfactory hairs, where proteins of the sensillum lymph and receptive dendrites are present. In control experiments with purified binding proteins, the proportion of pheromone bound to the oxidized PBP did not increase with increasing incubation time, suggesting that disulfide formation does not occur spontaneously but is mediated by the sensory hairs, possibly by interaction with the receptor cell membrane. These data suggest that arriving hydrophobic pheromone molecules are first bound by the reduced PBP and transported through the aqueous sensillum lymph towards the receptor molecules of the dendritic membrane. The oxidized complex might not be able to activate further receptors and, thus, effectively deactivate the pheromone molecules within the sensillum lymph.

• Schmidt FS, Skerra A
• The bilin-binding protein of Pieris brassicae. cDNA sequence and regulation of expression reveal distinct features of this insect pigment protein.
• Eur J Biochem. 1994; 219: 855-63
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• The bilin-binding protein (BBP) is a blue pigment protein which is abundant in the butterfly Pieris brassicae. In an attempt to clarify the physiological role of this member of the lipocalin family of proteins, its complete cDNA was cloned and expression of the BBP gene in P. brassicae was investigated. It was found that synthesis of the BBP mRNA is highly regulated during the insect's ontogenesis. In larvae after the third ecdysis as well as in pupae and adults, large amounts of the mRNA are present. Each of these stages itself displays a distinct time course of mRNA synthesis. In addition, BBP is expressed tissue specifically, with the fat body being the major source of this secretory protein in the larvae. Hence, the expression pattern of BBP in this organism is markedly different from the closely related pigment protein insecticyanin in Manduca sexta. Finally, the bacterial expression of BBP in a functional state was established as a basis for the future analysis of its ligand-binding functions by protein engineering.

• Takahashi S, Inoue H, Fukui K, Miyake Y
• Structure and function of renin binding protein.
• Kidney Int. 1994; 46: 1525-7

• Magee J, Kraynack N, Massey HC Jr, Telfer WH
• Properties and significance of a riboflavin-binding hexamerin in the hemolymph of Hyalophora cecropia.
• Arch Insect Biochem Physiol. 1994; 25: 137-57
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• A riboflavin-binding hexamerin isolated from pupal hemolymph of Hyalophora cecropia has a native M(r) of 510,000, subunit M(r) of 85,000, and a 5% carbohydrate content. An intrachain cross-link was confirmed in protease limit digests. Ellman titration confirmed the presence of a sulfhydryl group, which is needed for this linkage. Though Cu2+ is known to promote the linkage, heavy metals were not detected in the isolate. Heat denaturation released ligand with the absorbency, fluorescence spectra, and chromatographic behavior of riboflavin. Binding resulted in substantial quenching of the fluorescence of both the isoalloxazine in riboflavin and of aromatic groups in the apoprotein. Kinetic analysis indicated a KD of 2.5 x 10(-7) M for riboflavin, 1.3 x 10(-7) M for lumiflavin, and greater than 1 x 10(-6) M for FMN and FAD. Over four moles of flavin were bound per mole of hexamerin. The amount of riboflavin in pupal hemolymph is sufficient to occupy only 2-3 of these sites. Riboflavin is also associated with lipophorin and vitellogenin, but the molar ratios after protein isolation were low. On a standard laboratory diet, riboflavin is in great excess, but most of it is apparently excreted before the apoprotein first appears in the hemolymph, just before wandering. The concentration of riboflavin-binding hexamerin rises to 15-30 mg/ml in pupae; relative to other hexamerins, very little is stored in the fat body. All of the apoprotein and 75% of riboflavin disappear from the hemolymph during adult development. An amount of flavin at least equal to that stored in pupal hemolymph is transferred to the eggs formed during this period.

• Du G, Ng CS, Prestwich GD
• Odorant binding by a pheromone binding protein: active site mapping by photoaffinity labeling.
• Biochemistry. 1994; 33: 4812-9
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• The bacterially expressed recombinant pheromone binding protein (PBP) of Antheraea polyphemus was photoaffinity labeled with (6E,11Z)-[3H]hexadecadienyl diazoacetate, a photoactivatable analog of the naturally occurring acetate pheromone. Radiolabeled peptides were separated from an endoproteinase Lys-C digestion by HPLC and characterized by Edman degradation. The label was exclusively found in the Asp39-Lys58 fragment. Cleavage of this peptide (DDYVMTDRLAGCAINCLATK) with Arg-C gave a single radiolabeled peptide (DDYVMTDR), which was predicted to be alpha-helical. The adjoining LAGCAINCLATK fragment, which is highly conserved in PBP sequences, was predicted to be a hydrophobic beta-strand and has been proposed to be important in recognition of the alkadienyl chain. Edman degradation confirmed the location of the covalently attached ligand at Thr44 of the smaller hydrophilic peptide. In addition, the synthesis of the newly identified pheromone component (4E,9Z)-tetradecadienyl acetate and its photoaffinity analog, (4E,9Z)-[3H]tetradecadienyl diazoacetate, is also described. Mapping of PBP photoaffinity labeled by (4E,9Z)-[3H]14:Dza revealed that the hydrophobic region Asp21-Lys38 adjacent to the primary binding domain Asp39-Lys58 contained a second modification site. The 14-carbon odorant molecule thus had two binding positions within the recognition site, while only a single binding position was available to the 16-carbon pheromone.

• Prestwich GD
• Bacterial expression and photoaffinity labeling of a pheromone binding protein.
• Protein Sci. 1993; 2: 420-8
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• The first high-level production of a binding-active odorant binding protein is described. The expression cassette polymerase chain reaction was used to generate a DNA fragment encoding the pheromone binding protein (PBP) of the male moth Antheraea polyphemus. Transformation of Escherichia coli cells with a vector containing this construct generated clones which, when induced with isopropyl beta-D-thiogalactopyranoside, produced the 14-kDa PBP in both the soluble fraction and in inclusion bodies. Purification of the soluble recombinant PBP by preparative isoelectric focusing and gel filtration gave > 95% homogeneous protein, which was immunoreactive with an anti-PBP antiserum and exhibited specific, pheromone-displaceable covalent modification by the photoaffinity label [3H]6E,11Z-hexadecadienyl diazoacetate. Recombinant PBP was indistinguishable from the insect-derived PBP, as determined by both native and denaturing gel electrophoresis, immunoreactivity, and photoaffinity labeling properties. Moreover, the insoluble inclusion body protein could be solubilized, refolded, and purified by the same procedures to give a recombinant PBP indistinguishable from the soluble PBP. Proton NMR spectra of the soluble and refolded protein provide further evidence that they possess the same folded structure.

• Bryan J, Edwards R, Matsudaira P, Otto J, Wulfkuhle J
• Fascin, an echinoid actin-bundling protein, is a homolog of the Drosophila singed gene product.
• Proc Natl Acad Sci U S A. 1993; 90: 9115-9
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• A cDNA for fascin, an actin-bundling protein in echinoderms, has been cloned, sequenced, and expressed. The predicted mass of the protein is approximately 55 kDa, similar to that observed for fascin purified from sea urchin eggs. Bacterially expressed fascin reacts with antibodies prepared against sea urchin egg fascin. Fascin has a strong sequence similarity to the singed gene (sn) product in Drosophila and has similarities with a 55-kDa human actin-bundling protein. No extensive similarities were found with other known actin-binding/bundling proteins, indicating that this is a separate gene family.

• Krieger J, Raming K, Prestwich GD, Frith D, Stabel S, Breer H
• Expression of a pheromone-binding protein in insect cells using a baculovirus vector.
• Eur J Biochem. 1992; 203: 161-6
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• A cDNA encoding a pheromone-binding protein from the male silkmoth Antheraea pernyi has been integrated into the genome of the Autographa californica multiple nuclear polyhydrosis virus such that the transcription was under the control of the strong polyhedrin promoter. Recombinant pheromone-binding protein was expressed in a baculovirus-infected insect cell line (Sf9) and secreted from the cells into the culture medium. Using a two-step protocol, recombinant pheromone-binding protein has been isolated and purified to homogeneity. Pheromone binding of recombinant protein has been demonstrated using a tritiated analog of (E,Z)-6,11-hexadecadienyl acetate.

• Krieger J, Raming K, Breer H
• Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity.
• Biochim Biophys Acta. 1991; 1088: 277-84
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• Genomic DNA from the silk moth Antheraea pernyi bearing the gene of a pheromone binding protein has been isolated from a partial genomic library using specific cDNA probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene. A DNA fragment of a second gene was isolated and the complete primary structure of a corresponding cDNA clone was unravelled. The expression of two different genes, giving rise to different pheromone binding proteins, implies a more specific function of these proteins than was hitherto assumed.

• Vogt RG, Rybczynski R, Lerner MR
• Molecular cloning and sequencing of general odorant-binding proteins GOBP1 and GOBP2 from the tobacco hawk moth Manduca sexta: comparisons with other insect OBPs and their signal peptides.
• J Neurosci. 1991; 11: 2972-84
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• Odorant-binding proteins (OBPs) are small, water-soluble proteins uniquely expressed in olfactory tissue of insects and vertebrates. OBPs are present in the aqueous fluid surrounding olfactory sensory dendrites and are thought to aid in the capture and transport of hydrophobic odorants into and through this fluid. OBPs may represent the initial biochemical recognition step in olfaction, because they transport odorants to the receptor neurons. Insect OBPs are represented by multiple classes: pheromone-binding proteins (PBPs) and general odorant-binding proteins (GOBP1 and GOBP2). PBPs associate with pheromone-sensitive neurons, while GOBPs associate with general odorant-sensitive neurons. Analysis of N-terminal amino acid sequences of 14 insect OBPs isolated from six species indicated that the PBPs were variable and the GOBPs were highly conserved. However, inferred properties of these proteins were based only on partial sequence data. We now report the full-length sequences of a GOBP1 and GOBP2 from the moth Manduca sexta and compare these sequences with those of PBPs from three species, including M. sexta, Antheraea polyphemus, and A. pernyi. We also compare these with a GOBP2 of A. pernyi, previously identified only as a novel OBP. These comparisons fully support our N-terminal analysis. The signal peptide sequences of seven insect OBPs reveal conserved sequences within OBP classes, but not between OBP classes even within the same animal species. This suggests that multiple OBPs may be coexpressed in the same cell type, but differentially processed in a class-specific manner. Properties of the GOBPs suggest that general olfaction is broadly receptive at the periphery. Properties of the PBPs suggest that pheromone olfaction is discriminatory at the periphery, and that the initial biochemical steps in pheromone detection may play an active role in odor perception.

• Peitsch MC, Boguski MS
• Is apolipoprotein D a mammalian bilin-binding protein?
• New Biol. 1990; 2: 197-206
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• Human apolipoprotein D (APO-D) is a serum glycoprotein that has no sequence similarity with other apolipoproteins but rather belongs to the alpha 2-microglobulin superfamily whose other members transport small hydrophobic ligands in a wide variety of biological contexts. To investigate the ligand specificity of APO-D, we analyzed its relationship with the other members of this superfamily and constructed a detailed molecular model using the atomic coordinates of its most closely related homolog--insecticyanin from the tobacco hornworm, Manduca sexta. We studied the geometry of the binding pocket of APO-D and the topology of characteristic patches of both hydrophobic and polar side chains that also occur in crystal structures of insecticyanin and bilin-binding protein from the butterfly Pieris brassicae. From the data obtained we hypothesize that heme-related compounds may be more favorable ligands for APO-D than either cholesterol or cholesteryl ester. Preliminary experiments showed that purified human APO-D binds bilirubin in an approximately one-to-one molar ratio. These results suggest a new biological role for APO-D that is more congruent with its tissue distribution and evolutionary history.

• Raming K, Krieger J, Breer H
• Primary structure of a pheromone-binding protein from Antheraea pernyi: homologies with other ligand-carrying proteins.
• J Comp Physiol [B]. 1990; 160: 503-9
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• An antennal cDNA clone encoding the complete sequence (163 amino acids) of a pheromone-binding protein precursor from the male silk moth, Antheraea pernyi, was isolated using oligonucleotide probes. The cloned cDNA was expressed and the translation product detected by specific antibodies. The deduced protein sequence consists of a signal peptide of 21 amino acids and a mature binding protein of 142 amino acid residues. The predicted structure of this protein is homologous to binding-proteins from different insect species which have previously been identified, but shows no similarities to odorant-binding proteins from vertebrates, suggesting that soluble odorant-binding proteins in insects and vertebrates represent an evolutionary convergence.

• Hayashi H, Nakano M, Shibanaka Y, Fujita N
• Expression of a silkworm eclosion hormone gene in yeast.
• Biochem Biophys Res Commun. 1990; 173: 1065-71
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• Recombinant silkworm eclosion hormone was produced for the first time in yeast which was transformed with a shuttle plasmid containing a construct coding a signal peptide and the mature sequence of the silkworm eclosion hormone. Successfully transformed yeast processed recombinant silkworm eclosion hormone I (EH-I) and transported it to periplasm at the concentration of 60 micrograms per liter of culture. The biological activity of the purified recombinant silkworm eclosion hormone exhibited the ED50 value of 0.2 ng which is the same as that of the authentic hormone isolated from the silkworm brain.

• Vogt RG, Kohne AC, Dubnau JT, Prestwich GD
• Expression of pheromone binding proteins during antennal development in the gypsy moth Lymantria dispar.
• J Neurosci. 1989; 9: 3332-46
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• We have identified 2 olfactory specific proteins in the gypsy moth Lymantria dispar that are uniquely associated with the male antennae, the principal olfactory organs of this animal. These proteins were the major soluble protein components of the olfactory sensilla, present in equivalent amounts. Both proteins comigrated on SDS-PAGE, showing an apparent molecular mass of 15,000 Da but migrated separately on non-SDS-PAGE, indicating differences in net charge. N-terminal amino acid sequence analysis showed that the 2 proteins share 50% identity, indicating that they are genetically distinct homologs. Both proteins bound the L. dispar sexpheromone, associated with antisera prepared against the previously identified phermone-binding protein (PBP) of the moth Antheraea polyphemus, and shared sequence identity with the A. polyphemus PBP. These 2 proteins are therefore identified as L. dispar PBPs and are termed PBP1 and PBP2 based on their migration differences on non-SDS-PAGE. It is estimated that PBP1 and PBP2 are present in the sensilla lumen at a combined concentration of 13.4 mM. The expression of the L. dispar PBPs was examined during the 11 d development of the adult antenna. PBP1 and PBP2 were first detected by non-SDS-PAGE analysis and Coomassie blue staining 3 d before adult eclosion, on day A-3. Levels increased, reaching a plateau on day A-1 that continued into adult life. In vivo labeling studies indicated that the rate of PBP synthesis increased from A-3 to a plateau on A-2, where it remained into adult life. In vitro translations of antennal mRNAs indicated that translatable PBP mRNA was available at a very low level on day A-4, increased slightly on A-3 and dramatically on A-2, and remained at a high level into adult life. PBP mRNA represented the major translatable mRNA in the antenna during this period. It was estimated that the PBPs undergo a combined steady-state turnover of 8 x 10(7) molecules/hr/sensillum. Cursory in vivo and in vitro translation studies of antennal mRNA from A. polyphemus and Manduca sexta showed similar temporal patterns of PBP expression, suggesting that the L. dispar observations are general.

• Gyorgyi TK, Roby-Shemkovitz AJ, Lerner MR
• Characterization and cDNA cloning of the pheromone-binding protein from the tobacco hornworm, Manduca sexta: a tissue-specific developmentally regulated protein.
• Proc Natl Acad Sci U S A. 1988; 85: 9851-5
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• cDNA encoding pheromone-binding protein (PBP), the major soluble protein in olfactory sensilla of male moths, has been cloned from the tobacco hornworm, Manduca sexta. A study of the developmental time course of PBP reveals that it is first synthesized just prior to eclosion and that the percentage of antennal mRNA encoding PBP shifts from zero to about 20% at that time. PBP is also found in sensilla from female M. sexta antennae. No amino acid sequence homology is observed between PBP and the vertebrate odorant-binding protein.