title: 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.
abstract: In 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 (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa3-Cys) 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 (Schistocerca gregaria 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 in vitro. 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 in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes 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.
authors.affiliation: Laboratory of Developmental Physiology, Genomics and Proteomics, Catholic University of Leuven, Naamsestraat 59, B-3000, Leuven, Belgium. gert.simonet@bio.kuleuven.ac.be
authors.forename: Gert
authors.initials: G
authors.lastname: Simonet
authors.forename: Bert
authors.initials: B
authors.lastname: Breugelmans
authors.forename: Paul
authors.initials: P
authors.lastname: Proost
authors.forename: Ilse
authors.initials: I
authors.lastname: Claeys
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Damme
authors.forename: Arnold
authors.initials: A
authors.lastname: De Loof
authors.forename: Jozef
authors.initials: J
authors.lastname: Vanden Broeck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D006110:Grasshoppers / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D019476:Insect Proteins; D020033:Protein Isoforms; D011506:Proteins; C106709:pacifastin