Potential Cooperations between Odorant-Binding Proteins of the Scarab Beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae)

It was previously thought that the odorant binding proteins (OBPs) in the sensillum lymph might serve as carriers, which could carry lipophilic odorant molecules to olfactory receptors. In this study, two novel OBP genes of the scarab beetle Holotrichia oblita were screened using an antennal cDNA library. The full cDNA of HoblOBP3 and HoblOBP4 was cloned using reverse transcription PCR and rapid amplification of the cDNA ends. Homology modeling of both OBPs was performed using SWISS-MODEL on-line tools. Next, the two OBPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. The ligand-binding properties of HoblOBP3 and HoblOBP4 in 42 ligands respectively were measured using the fluorescence probe N-phenyl-naphthylamine (1-NPN). The results obtained from competitive binding assays demonstrated that HoblOBP4 showed a broader range of binding affinities to the test compounds, while HoblOBP3 displays more specific binding affinity. Furthermore, other OBPs and CSPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. Binding curves were measured for binary mixtures of OBPs and CSPs using 1-NPN, and the Scatchard plots exhibited "J"-like nonlinear correlation trends in some samples. In addition, competitive binding assays of the HoblOBP1 and HoblOBP2 mixtures and of the HoblOBP2 and HoblOBP4 mixtures with representative compounds unexpectedly demonstrated good affinity, which revealed extreme differences that were only obtained using the individual proteins. In the immunocytochemical analysis, colocalization of HoblOBP1 and HoblOBP2, and of HoblOBP2 and HoblOBP4, was detected in the sensilla basiconica and sensilla placodea, respectively. All of these results suggested that HoblOBP1 and HoblOBP2, as well as HoblOBP2 and HoblOBP4, may serve as heterodimers in the sensillum lymph.