Partial characterization of trypsin-like protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris.
Based on substrate specificity, an alkaline pH optimum, sensitivity to selected proteinase inhibitors, and molecular analysis, we provide evidence for the presence of a trypsin-like serine proteinase in the salivary gland complex (SGC) of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The predominant activity in extracts of the SGC against N(2)-benzoyl-L-arginine-p-nitroanilide (L-BApNA) was at pH 10, but a minor peak of activity also occurred at pH 5. The major BApNAase activity focused at 10.4 during preparative isoelectric focusing and was eluted with an apparent molecular weight of 23,000 from a calibrated gel filtration column. The BApNAase fraction gave a single major band when analyzed on a casein zymogram. The activity was completely suppressed by the serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor. A cDNA coding for a trypsin-like protein in the salivary glands of L. lineolaris was cloned and sequenced. The 971bp cDNA contained an 873-nucleotide open reading frame encoding a 291-amino acid trypsin precursor. The encoded protein included amino acid sequence motifs that are conserved with four homologous serine proteases from other insects. Typical features of the putative trypsin-like protein from L. lineolaris included the serine protease active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, the residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for trypsin like enzymes in the salivary glands of L. lineolaris.
Zeng
F
F
Biological Control and Mass Rearing Research Unit, USDA/ARS, P.O. Box 5367, Mississippi State, MS 39762, USA. fzeng@bcmrru.msstate.edu
Zhu
Y
Y
Cohen
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006430:Hemiptera / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000235:genetics*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012469:Salivary Glands / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D018076:DNA, Complementary; D019476:Insect Proteins; C114880:trypsin-like serine protease; D012697:Serine Endopeptidases