Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Abstract

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-α-benzoyl- l-arginine p-nitroanilide (BA pNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BA pNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BA pNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl- l-leucylamido-(4-guanidino) butane (E-64) inhibited BA pNAase activity by about 30%. BA pNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BA pNAase activity was primarily located in the midgut region with about 1.5-fold more BA pNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23–24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77–81% sequence identity. The three encoded trypsinogens shared 54–62% identity in their amino acid sequences and had 16–18 residues of signal peptides and 12–15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG 62–65, the catalytic amino acid triad of serine proteinase active sites (His 109, Asp 156, Ser 257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp 251, Gly 274, Gly 284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys 158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).