Odorant-binding proteins (OBPs) mediate both perception and release of semiochemicals in insects. These proteins are the ideal targets for understanding the olfactory code of insects as well as for interfering with their communication system in order to control pest species. The two sibling Lepidopteran species Helicoverpa armigera and H. assulta are two major agricultural pests. As part of our aim to characterize the OBP repertoire of these two species, here we focus our attention on a member of this family, OBP10, particularly interesting for its expression pattern. The protein is specifically expressed in the antennae of both sexes, being absent from other sensory organs. However, it is highly abundant in seminal fluid, is transferred to females during mating and is eventually found on the surface of fertilised eggs. Among the several different volatile compounds present in reproductive organs, OBP10 binds 1-dodecene, a compound reported as an insect repellent. These results have been verified in both H. armigera and H. assulta with no apparent differences between the two species. The recombinant OBP10 binds, besides 1-dodecene, some linear alcohols and several aromatic compounds. The structural similarity of OBP10 with OBP1 of the mosquito Culex quinquefasciatus, a protein reported to bind an oviposition pheromone, and its affinity with 1-dodecene suggest that OBP10 could be a carrier for oviposition deterrents, favouring spreading of the eggs in these species where cannibalism is active among larvae.
The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.
Odorant-binding proteins (OBPs) are soluble proteins, whose role in olfaction of insects is being recognized as more and more important. We have cloned, expressed and purified an OBP (HarmOBP7) from the antennae of the moth Helicoverpa armigera. Western blot experiments indicate specific expression of this protein in the antennae of adults. HarmOBP7 binds both pheromone components Z-11-hexadecenal and Z-9-hexadecenal with good affinity. We have also performed a series of binding experiments with linear aldehydes, alcohols and esters, as well as with other compounds and found a requirement of medium size for best affinity. The affinity of OBP7, as well as that of a mutant lacking the last 6 residues does not substantially decrease in acidic conditions, but increases at basic pH values with no significant differences between wild-type and mutant. Binding to both pheromone components, instead, is negatively affected by the lack of the C-terminus. A second mutant, where one of the three lysine residues in the C-terminus (Lys123) was replaced by methionine showed reduced affinity to both pheromone components, as well as to their analogues, thus indicating that Lys123 is involved in binding these compounds, likely forming hydrogen bonds with the functional groups of the ligands.
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