Functional analysis of TcCTLP-5C2, a chymotrypsin-like serine protease needed for molting in Tribolium castaneum

Insect Biochemistry and Molecular Biology
Daniel AlbaumHans Merzendorfer

Abstract

In a previous study, we have characterized a gene family encoding chymotrypsin-like proteases from the red flour beetle, Tribolium castaneum (TcCTLPs). We identified 14 TcCTLP genes that were predominantly expressed in the midgut, where they presumably function in digestion. Two genes (TcCTLP-6C and TcCTLP-5C2), however, additionally showed considerable expression in the carcass, and RNAi studies demonstrated that they are required for molting (Broehan et al., 2010; Insect Biochem. Mol. Biol 40, 274-83). Thus, the enzyme has distinct functions in different physiological environments. To study molecular adaptations that facilitate enzyme function in different environments, we performed an in-depth analysis of the molecular and enzymatic properties of TcCTLP-5C2. We expressed different mutated versions of TcCTLP-5C2 in form of factor Xa activatable pro-enzymes in insect cells using a baculoviral expression system, and purified the recombinant proteins by affinity chromatography. By measuring and comparing the enzyme activities, we obtained information about the significance of single amino acid residues in motifs that determine substrate specificity and pH tolerance. Further, we showed that TcCTLP-5C2 is modified by N-glycosylati...Continue Reading

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