N-glycosylation at non-canonical Asn-X-Cys sequence of an insect recombinant cathepsin B-like counter-defense protein

CmCatB, a cowpea bruchid cathepsin B-like cysteine protease, facilitates insects coping with dietary protease inhibitor challenge. Expression of recombinant CmCatB using a Pichia pastoris system yielded an enzymatically active protein that was heterogeneously glycosylated, migrating as a smear of ≥ 50 kDa on SDS-PAGE. Treatment with peptide:N-glycosidase F indicated that N-glycosylation was predominant. CmCatB contains three N-glycosylation Asn-X-Ser/Thr consensus sequences. Simultaneously replacing all three Asn residues with Gln via site-directed mutagenesis did not result in completely unglycosylated protein, suggesting the existence of additional atypical glycosylation sites. We subsequently investigated potential N-glycosylation at the two Asn-X-Cys sites (Asn100 and Asn236) in CmCatB. Asn to Gln substitution at Asn100-X-Cys on the background of the double mutation at the canonical sites (m1m2, Asn97→Gln and Asn207→Gln) resulted in a single discrete band on the gel, namely m1m2c1 (Asn97→Gln, Asn207→Gln and Asn100→Gln). However, another triple mutant protein m1m2c2 (Asn97→Gln, Asn207→Gln and Asn236→Gln) and quadruple mutant protein m1m2c1c2 were unable to be expressed in Pichia cells. Thus Asn236 appears necessary for protein expression while Asn100 is responsible for non-canonical glycosylation. Removal of carbohydrate moieties, particularly at Asn100, substantially enhanced proteolytic activity but compromised protein stability. Thus, glycosylation could significantly impact biochemical properties of CmCatB.