Patricia Nagnan-Le Meillour

Pheromone binding proteins of the moth Mamestra brassicae: Specificity of ligand binding

Several isoforms of pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP) were previously characterized in the antennae of the cabbage armyworm Mamestra brassicae L. (Lepidoptera: Noctuidae). In further investigations, we used two-dimensional electrophoresis and Western-blotting with antibodies raised against the PBPs of the male: this method revealed more proteins with molecular weight and isoelectric points similar to those of OBPs and confirmed the high level of microdiversity suspected for this family of proteins. The binding of the tritiated major pheromone compound, Z11-16:Ac, with male and female antennal extracts and purified PBPs from male antennae was studied. Only the two isoforms Mbra-1 and Mbra-1′ (N-terminus: SKELI) bound the labelled pheromone, whereas no binding was observed with the Mbra-2 (N-terminus: SQEIM). In female antennal extracts, binding was shown between Z11-16:Ac and the proteins Mbra-1 and GOBP2. These results constitute an unambiguous demonstration of the binding specificity of a PBP to a pheromonal ligand, supporting the hypothesis of active participation of PBPs in odor discrimination, as a filter for odorants, prior to the receptor activation.

Purification and characterization of multiple forms of odorant/pheromone binding proteins in the antennae of mamestra brassicae (noctuidae)

Proteins extracted form the antennae of Mamestra brassicae (L.) (Lepidoptera: Noctuidae) adults were biochemically characterized as pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP). PBP and GOBP were purified by two successive and different HPLC (high performance liquid chromatography) systems and native polyacrylamide gel electrophoresis (native-PAGE). Their N-terminal sequence was determined by Edman microsequencing. The combined results showed evidence for three different PBPs in males, and two different PBPs in females. In addition, one GOBP was characterized in both males than in females antennae. In the males, two isoforms of PBP have the same N-terminal sequence, but different apparent mobilities and hydrophobicities: they could be separated by electrophoresis and reverse phase-HPLC (RP-HPLC). The other PBP sequence (SQEIM) showed particularly high homology (88%) with the PBP of Heliothis virescens, another noctuid moth. The existence of several forms of PBP in the same animal strongly supports the hypothesis of the specificity of binding between the proteins and their odorant ligands, the pheromonal compounds. The observed microdiversity at the soluble proteins level could provide a good model for studying their involvement in the initial stages of odor discrimination.

Molecular cloning of two pheromone binding proteins in the cabbage armyworm Mamestra brassicae

Two cDNA clones encoding pheromone binding proteins (PBPs) were isolated from antennal cDNA of Mamestra brassicae by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends-PCR (RACE-PCR) performed with specific primers deduced from the N-terminal sequences of two PBPs previously reported. The deduced protein sequences of the two PBPs showed a strong relationship between primary structures and functional properties of the corresponding mature proteins.

Primary culture of antennal cells of Mamestra brassicae: morphology of cell types and evidence for biosynthesis of pheromone-binding proteins in vitro.

Abstract.A culture technique for the in vitro growth and differentiation of antennal cells of Mamestra brassicae is described. The morphology of the growing cells is described. At least five morphological cell types can be distinguished in the cultures. Neuronal cells (type A) were characterized morphologically and immunocytochemically with anti-HRP staining and could be separated into two subtypes, A1 and A2, on the basis of the size of the cell body. Non-neuronal cells could be divided into four types. Type-B cells consisted of large and flat cells with a veil-like cytoplasm. They usually adhered to each other and formed groups of 20 to 50 cells. Type C were large and flat cells, highly variable in size and shape. They closely resembled insect glial cells in culture. Type-D cells grew thick processes, up to 100 μm long. These elongated processes resembled trichoid hairs, and D cells are most probably trichogen cells. Type E were spindle-shaped cells present in low number in the cultures. A high proportion of similar cells in the hemolymph of pupae suggests that E cells are hemocytes. Pheromone-binding proteins were detected using specific antisera in the medium of 3- to 4-week-old cultures.

Biochemistry and diversity of insect odorant-binding proteins

Publisher Summary Odorant-binding proteins (OBPs) are abundant in the sensillar lymph of insect antennae and participate in olfactory perireceptor events, such as transport of the hydrophobic odorant through the aqueous medium, presentation of the odor to olfactory receptors, and deactivation of the signal. This chapter describes the biochemistry and molecular biology of pheromone detection in the noctuid moth, Mamestra brassicae. The OBPs are diverse ranging from numerous species of several insect orders including Lepidoptera, Diptera, Coleoptera, and Hymenoptera, Hemiptera, and Phasmatodea. Classification defines OBP-Type 1 and OBP-Type 2 based on phylogeny, tissue localization, and structural features. The advantage of the functional approach to characterize the diversity of OBPs inside species is characterized. The construction of EST antennal libraries with a high EST number to be representative of the sequence diversity has identified not only new OBPs, but also SAPs, pheromone-degrading enzymes, sensory-neuron-membrane-protein-like sequences, and other elements involved in the pheromone transduction process.