STRUCTURE AND FUNCTION RELATIONSHIPS IN INSECT ODORANT BINDING PROTEINS: LIGAND SPECIFICITY AND ROLE IN ODOR DISCRIMINATION

Patricia NAGNAN-LE MEILLOUR, MAIBECHE Martine, JACQUiN-JOLY Emmanuel le, MAI DA Rosario, BLAIS+ Jean-Claude, TABET+ Jean-Claude, DESCOINS Charles
INRA, Unité de Phytopharmacie et des Médiateurs Chimiques, Route de Saint-Cyr, F-76026 Versailles CEDEX FRANCE.
+Université Pierre et Marie Curie, Laboratoire de chimie structurale organique et biologique, CNRS EP 103, 4 place Jussieu, F-75252 PARIS CEDEX 05 FRANCE (nagnan@versailles.inra.fr)

The pheromonal system of the noctuid moth Mamestra brassicae is well known since three receptor cells were characterized-1, responding to the major compound of the pheromone (Z11 -16:Ac) and different antagonists used by heterospecific species (Z9-14:Ac, Z11-16:Ald). At a biochemical point of view, we identified and purified at least three pheromone-binding proteins (PBPs: Mbra-1, Mbra-2 and Mbra-3) and one general odorant-binding protein (GOBP2) from the male and female antennae-2

We developped monoclonal antibodies raised against Mbra-1 and Mbra-2 to localize eventual PBP-like proteins in other insect species using Z9-14:Ac-3. We chracterized the PBPs of Spodoptera descoinsi and S. IaRfascia, which share 75% homology in N-terminal sequence with M brassicae (88% between H virescens-4 and M. brassicae).

The number and the molecular weight of OBPs were precised by MALDI- TOF-MS measurements after electroblotting onto PVDF membranes-5. The presence of numerous other OBP molecules, different in size and in N-terminal sequence are suspected in antennal extracts. Such a microdiversity in OBPs suggests a high specificity towards the odorant ligand.

Binding-on-gel experiments showed specific binding between PBPs-Mbra and pheromone compounds6. Particularly, the Z11-16:Ac, which activity can alone elicit males attractivity is only bound by the PBP-Mbra-1. In addition, PBP-Mbra-2 showed high N-terminal homology with PBPs of Spodoptera spp and Heliothis spp, other noctuid moths using antagonists for M. brassicae. This argument strongly supports the hypothesis that the function of the PBP, in term of ligand specihcity, is encoded in its primary structure-7

We are now able to associate a pheromonal compound (Z11-16:Ac) with a PBP (Mbra-1) and an olfactory receptor neuron type (cell A). Development of cell cultures of ORNs from M. brassicae, expressing specific PBPs provides a good << in vitro >> model to study the key-level of the olfactory transduction, i.e.: the activation of the olfactory receptors by specific complexes PBP/pheromone. in addition, after cloning of PBP-Mbra1 gene and GOBP2 gene, their expression is in progress in baculovirus vector for further structural studies (cristallography and NMR).

Literature
  1. Renou M., Lucas P.: J. Insect Physiol. 40, 75 (1994)
  2. Nagnan-Le Meillour P., Huet J. C., Maïbèche M., Pernollet J. C., Descoins C.: Insect Biochem. Molec. Biol. 26, 59 (1996)
  3. Nagnan-Le Meillour P., Blais J. C., Jacquin-Joly E., Maïbèche M., Tabet J. C., Descoins C.: submitted to Insect Biochem. Molec. Biol.
  4. Krieger J., Ganble H., Ramins K., Breer H.: Insect Biochem. Molec. Biol. 23, 449 (1993)
  5. Blais J C., Nagnan-Le Meillour P., Bolbach G., Tabet J. C.: R. Com. Mass Spectrom. 10, 1(1996)
  6. Maïbèche M., Sobrio F., Letterre M., Dubroca J., Jacquin-Joly E., Nagnan-Le Meillour P.: submitted to Insect Biochem. Molec. Biol.
  7. Du G., Prestwich G. D.: Biochemistry 34, 8726 (1995)

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