Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria

Abstract

Soluble low-molecular-mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40±50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29±Cys38 and Cys57±Cys60) and indicated the absence of any additional post-translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO 2 -sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera.Keywords: chemosensory proteins; contact sensilla; disulfide bridges; Schistocerca gregaria; sequence analysis.Locusts and grasshoppers are major pests in agriculture. They have a solitary and a gregarious phase, characterized by different behaviour and morphological features [1]. All crop damage is caused by individuals in the gregarious phase.In the species Schistocerca gregaria, the shift from the solitary to the gregarious phase is preceded by an associating phase, triggered by volatile aromatic compounds, such as guaiacol, veratrol and phenylacetonitrile [2]. In the gregarious phase, the same chemicals, and perhaps related structures, induce aggregation of great numbers of individuals. Under these conditions this species becomes a plague and can destroy entire crops. Therefore, it is evident that these insects rely on chemical communication and that their populations could be controlled by the use of the appropriate chemical stimuli.Despite such pressing objectives, biochemical study of the olfactory system had been limited until recently to Lepidopteran species with large antennae. In the last few years, however, molecular biology techniques have made such research feasible in small insects of wider interest, such as Drosophila melanogaster. In this paper we report the isolation of soluble low-molecularmass proteins in antennae, tarsi and labrum of S. gregaria, their complete structural characterization by combined Edman degradation/MS procedures and the cloning of...

Moth chemosensory protein exhibits drastic conformational changes and cooperativity on ligand binding

“…C hemosensory proteins (CSPs) have been identified in antennae from Drosophila melanogaster and in antennae, proboscis, tarsi, and labrum from several insects (1)(2)(3)(4)(5)(6)(7). Several CSPs from antenna or proboscis have also been isolated in the moth Mamestra brassicae (3).…”

“…Several CSPs from antenna or proboscis have also been isolated in the moth Mamestra brassicae (3). Due to their localization, these proteins have been proposed to be involved in chemical signal transduction or in chemo-perception, either olfaction or taste (2)(3)(4)(5)(6). They might transport chemicals from air to receptors through the aqueous medium surrounding them, but their exact physiological roles have still to be assigned.…”

“…CSPs are shorter (110-115 aa) than the other classes of odorant transport proteins, pheromonebinding proteins (PBPs) or general odorant binding proteins (8) and share no sequence homology with them. They contain only four conserved cysteines forming two disulfide bridges (6) instead of three in the other classes. The structure of the PBP from Bombyx mori has been solved, revealing a 6 ␣-helical protein that undergoes large conformational changes on pH shift (9,10).…”

X-ray Structure and Ligand Binding Study of a Moth Chemosensory Protein

“…A third class of small proteins (average M r 13,000) has been identified in antennae from Drosophila melanogaster and in antennae and several sensorial organs (tarsi, labrum) from a wide range of species of the insect order (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). These proteins have been proposed to be involved in CO 2 detection (3), in chemical signal transmission in regenerating legs (5), or in chemo-perception (either olfaction or taste (9,12)), and they were therefore called chemosensory proteins (CSPs). CSPs are shorter (110 -115 amino acids) than PBP or GOBP, contain only 4 conserved cysteines forming two disulfide bridges (9), and share no sequence homology with them.…”

“…These proteins have been proposed to be involved in CO 2 detection (3), in chemical signal transmission in regenerating legs (5), or in chemo-perception (either olfaction or taste (9,12)), and they were therefore called chemosensory proteins (CSPs). CSPs are shorter (110 -115 amino acids) than PBP or GOBP, contain only 4 conserved cysteines forming two disulfide bridges (9), and share no sequence homology with them. They may also play a role in the transport of hydrophobic chemicals (volatile or not) from air or water to olfactory or taste receptors in a similar way as other transport proteins such as GOBPs or PBPs.…”