Cloning and characterization of chymotrypsin- and trypsin-like cDNAs from the gut of the Hessian fly [Mayetiola destructor (Say)].
Fifteen unique cDNA clones encoding trypsin- or chymotrypsin-like proteins were cloned and characterized from a gut cDNA library derived from Hessian fly [Mayetiola destructor (Say)] larvae. Based on sequence similarities, the cDNAs were sorted into five gene groups, which were named MDP1 to MDP5. Two of the gene groups, MDP1 and MDP2, encoded chymotrypsin-like proteins; the other three encoded putative trypsins. All deduced proteins have conserved His(87), Asp(136), and Ser(241) residues for the catalytic triad and three pairs of cysteine residues for disulfide bridge configurations. The substrate specificity determination residue at position 235 was also conserved in the putative trypsins and chymotrypsins. In addition, all the deduced protein precursors had a typical secretion signal peptide and activation peptide. Northern blot analysis revealed that all these gene groups were exclusively expressed in the larval stage. The expression profiles for each gene group differed significantly in different ages of the larva, as well as in different tissues. Protease activity analysis of gut extract, using specific inhibitors, demonstrated that serine proteases were the major digestive enzymes in the gut of M. destructor larvae. Serine protease inhibitors inhibited as much as 90% proteolytic activities of gut extract, whereas inhibitors specific to other proteases, including cysteine proteases, aspartic proteases, and metallo-proteases, inhibited only 10-24% of gut protease activity.
Zhu
Yu Cheng
YC
USDA-ARS-JWDSRC, PO Box 346/141 Exp Stn Rd, Stoneville, MS 38776, USA.
Liu
Xiang
X
Maddur
Ashoka A
AA
Oppert
Brenda
B
Chen
Ming-Shun
MS
D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000737:chemistry / Q000235:genetics*; D004064:Digestive System / Q000201:enzymology; D004175:Diptera / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016415:Sequence Alignment; D014357:Trypsin / Q000737:chemistry / Q000235:genetics*
D002918:Chymotrypsin; D014357:Trypsin