Effect of N-linked glycosylation in activity of CmCatB protein

Callosobruchus maculatus cathepsin B (CmCatB) is a counter-defense protein belonging to the papain family of cysteine proteases. We reported in our previous study that recombinant CmCatB expressed in yeast Pichia pastoris showed smear bands because of heterogeneous glycosylation and had cathepsin B enzymatic activity. CmCatB contains three typical N-linked glycosylation sites (N-X-S/T) and two atypical N-linked glycosylation sites (N-X-C). To investigate effect of N-linked glycosylation on activity of CmCatB protein, we attempted to express and purify recombinant proteins of non-glycosylated mutants by site-directed mutagenesis to change N into Q in the potential N-linked glycosylation sites (M1; N97Q, M2; N207Q, M3; N111Q, C1; N100Q; C2; N236Q). Recombinant CmCatB, CmCatB-M1M2, CmCatB-M1M2M3, and CmCatB-M1M2C1 were successfully expressed, although the yield of purified CmCatB-M1M2Cl was relatively low. Only recombinant CmCatB-M1M2C1 protein showed as a clear-cut band on SDS-PAGE indicating the existence of atypical N-inked glycosylation in CmCatB. Proteolytic activity of recombinant CmCatB mutant proteins were measured with rGSII-D88N protein as substrate at different pH conditions and time intervals, both recombinant CmCatB-M1M2 and CmCatB-M1M2C1 exhibited highest activity at pH4.5, same as their parental CmCatB. Recombinant CmCatB-M1M2C1 protein degraded rGSII-D88N protein faster than CmCatB and CmCatB M1M2, suggesting that N-glycosylation at this atypical site affects enzymatic activity of CmCatB protein.