Purification of pimplin, a paralytic heterodimeric polypeptide from venom of the parasitoid wasp Pimpla hypochondriaca, and cloning of the cDNA encoding one of the subunits.
We have previously detected a paralytic factor in gel filtration-separated venom from the endoparasitoid wasp Pimpla hypochondriaca which is active against the fly Musca domestica. Now we have further purified this factor, which we have called pimplin, by reverse phase chromatography, and established using SDS-PAGE that it has a molecular mass of approximately 22 kDa. A 40 ng dose of pimplin administered to adult M. domestica by intrahaemocoelic injection was sufficient to kill all flies tested. Treatment of pimplin with beta-mercaptoethanol prior to SDS-PAGE analysis resulted in the appearance of two polypeptides of approximately 15 and 6 kDa, indicating that pimplin is a heterodimer whose polypeptides are linked through a disulphide bond. Subunit masses of 10.544 and 6.318 kDa were determined using MALDI-TOF analysis indicating that the larger subunit migrates anomalously in SDS-PAGE. Using an oligonucleotide probe designed from N-terminal sequence obtained for the 15 kDa polypeptide, we have isolated a cDNA (pim1) encoding this larger pimplin subunit. The N-terminal amino acid sequence of pim1 occurred 28 residues beyond a predicted signal peptide cleavage site, indicating that pim1 is synthesised as a pre-propolypeptide which is secreted and proteolytically cleaved to yield the mature polypeptide stored in the venom sac. Beginning at the fourth residue of the mature pim1 venom polypeptide is a stretch of 46 residues consisting of alternating prolines, the significance of which is discussed in terms of possible host processing.
Parkinson
N
N
Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK. n.parkinson@csl.gov.uk
Smith
I
I
Audsley
N
N
Edwards
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D006793:Houseflies / Q000472:pathogenicity; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000235:genetics; D014862:Wasp Venoms / Q000235:genetics*; D014863:Wasps / Q000235:genetics* / Q000469:parasitology
D017931:DNA Primers; D018076:DNA, Complementary; D019476:Insect Proteins; D021122:Protein Subunits; D014862:Wasp Venoms; C471288:pimplin protein, Pimpla hypochondriaca