Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae).
Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.
Zeng
Fanrong
F
Biological Control and Mass Rearing Research Unit, USDA/ARS, P.O. Box 5367, Mississippi State, MS 39762, USA. fzeng@bcmrru.msstate.edu
Zhu
Yu Cheng
YC
Cohen
Allen C
AC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006430:Hemiptera / Q000201:enzymology* / Q000235:genetics; D019476:Insect Proteins / Q000235:genetics*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010802:Phylogeny; D012469:Salivary Glands / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000145:classification / Q000235:genetics*; D014357:Trypsin / Q000145:classification / Q000235:genetics*
D018076:DNA, Complementary; D019476:Insect Proteins; D012697:Serine Endopeptidases; D014357:Trypsin