Laboratory of Chemical Prospecting, National Institute of Sericultural and Entomological Science, 1-2 Ohwashi, Tsukuba 305-8634, Japan.
Pheromone binding proteins (PBPs) participate in the early olfactory processing of chemical signals in insect antennae by carrying the airborne hydrophobic pheromone molecules to the olfactory receptors, which are believed to be located in the dendritic surfaces of olfactory receptor neurons. Disulfide bond formation is the only posttranslational modification of these secretory proteins. In the moth PBPs, six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing was known about their linkage. 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. Supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN).