Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.
Two different classes of chemosensory proteins (CSPs) in Locusta migratoria have been identified on the basis of the molecular cloning of a series of different cDNAs from the antennae of this insect. Several CSP isoforms have been purified and biochemically characterized from antennal and wing extracts, some of them corresponding to expression products predicted for the identified cDNAs. In wings, the nature of the main endogenous ligand binding to these proteins was determined as oleoamide by a gas chromatography-mass spectrometric approach. One of these isoforms has been expressed in a bacterial system with high yield and used in a fluorescent binding assay. Competitive binding experiments have indicated the presence of long-chain compounds among the best ligands.
We have identified, cloned and expressed a new chemosensory protein (CSP) in the desert locust Schistocerca gregaria belonging to a third sub-class of these polypeptides. Polyclonal antibodies stained a band of 14 kDa, as expected, in the extracts of antennae and palps of the adults, but not in the 4th and 5th instars. In the related species Locusta migratoria, instead, the same antibodies cross-reacted only with a band of apparent molecular mass of 35 kDa in the extract of 1st-5th instars, but not in the adults. The recombinant protein binds the fluorescent probe N-phenyl-1-naphthylamine, but none of the compounds so far reported as pheromones for S. gregaria. The expression of the odorant-binding protein (OBP) and of CSPs of sub-classes I and II was also monitored in antennae, tarsi, palpi, wings and other organs of solitary and gregarious locusts in their nymphal and adult stages. OBP was found to be antenna specific, where it is expressed at least from the 3rd instar in both solitary and gregarious locusts. CSPs, instead, appear to be more ubiquitous, with different expression patterns, according to the sub-class. Immunocytochemistry experiments revealed that OBP is present in the sensillum lymph of sensilla trichodea and basiconica, while CSP-I and CSP-III were found in the outer sensillum lymph of sensilla chaetica and in the sub-cuticular space between epidermis and cuticle of the antenna. Sensilla chaetica on other parts of the body showed the same expression of CSP-I as those on the antenna.
Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-I), and 1 -115 (VS-2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/ PAGE and mass spectrometry analysis. However, their hydrodynamic behaviours in size-exclusion chromatography corresponded to that of proteins with a larger size. SDSPACE analysis of VS-1 and VS-2 after cross-linking with disuccinimidyl suberate indicated that both polypeptides form dimers. VS-2 was almost entirely dimeric at > 4 pM, but rapidly converted to monomer after dilution to 70 nM. The rapid dimer-monomer transition of VS-2 after dilution could be part of a mechanism for regulating its activity and localising its action. Immunological studies of VS-1 have shown that residues 37-70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides. The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments.Keywords: chromogranin; vasostatin ; recombinant DNA ; monoclonal antibody; antigenic region.Chromogranin A is an acidic protein belonging to a family of regulated secretory proteins present in the electron-dense granules of many endocrine and neuroendocrine tissues [I -31. Human chromogranin A is a hydrophilic protein of 439 amino acids, characterised by several post-translational modifications, including glycosylation, sulfation and phosphorylation [ 1, 4, 51. Chromogranin A has been suggested to be a low-affinity highcapacity Ca' + -binding protein involved in hormone packaging within secretory granules and in the regulation of secretory granule biogenesis and function [6, 71. Moreover, chromogranin A has been proposed to represent a precursor of biologically active peptides that are released together with various hormones [8]. Accordingly, chromogranin A contains a high number of dibasic sites thought to be important for tissue-specific proteolytic processing and biological activity [9-121. For instance, residues 248 -293 were found to be similar to pancreastatin, a pancreatic peptide that inhibits insulin secretion [I 31, whereas residues 124-143, called chromostatin, inhibit secretion of catechol- amines from chromaffin cells (141. Stimulated retrogradely perfused bovine adrenal medullae release the chromogranin A fragments corresponding to amino acids 1-76 and 1-113. These fragments, named vasostatin I and 11, respectively, were found to be endowed with vasoinhibitory activity on human blood vessels [lS, 161. Studies aimed at investigating the structure and the function of chromogranin A and its ...
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