Characterization of two novel pacifastin-like peptide precursor isoforms in the desert locust (Schistocerca gregaria): cDNA cloning, functional analysis and real-time RT-PCR gene expression studies

摘要

pIn the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (bCys/b-Xaasub9–12/sub-bCys/b-Asn-Xaa-bCys/b-Xaa-bCys/b-Xaasub2–3/sub-Gly-Xaasub3–6/sub-bCys/b-Thr-Xaasub3/sub-bCys/b) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (iSchistocerca gregaria/i PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed iin vitro/i. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an iin silico/i datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, iAedes aegypti/i. As in other insect pacifastin-related precursors, the iAedes/i precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1–3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism./p