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

- The chymotrypsin-like protease TcCTLP-5C2 from Tribolium castaneum is glycosylated at N137.
- Different mutated versions of TcCTLP-5C2 were expressed as factor Xa activatable pro-enzymes in insect cells.
- Enzyme kinetics were determined and the specificities were compared using different substrates.
- N-glycosylation alters the catalytic properties of TcCTLP-5C2 through allosteric effects.

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-glycosylation at amino acid position N137, which lies opposite to the catalytic cleft. Comparison of the enzymatic properties of non-glycosylated and glycosylated TcCTLP-5C2 versions showed that N-glycosylation decreases Vmax (maximum velocity) and kcat (turnover) while leaving the Km (specificity) unchanged. Thus, we provide evidence that N-glycosylation alters catalytic behavior by allosteric effects presumably due to altered structural dynamics as observed for chemically glycosylated enzymes.

351