Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1975693 | Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology | 2017 | 8 Pages |
Abstract
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 â¥Â 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, Asn97âGln and Asn207âGln) resulted in a single discrete band on the gel, namely m1m2c1 (Asn97âGln, Asn207âGln and Asn100âGln). However, another triple mutant protein m1m2c2 (Asn97âGln, Asn207âGln and Asn236âGln) 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.
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Authors
Yong Hun Chi, Yoon Duck Koo, Susie Y. Dai, Ji-Eun Ahn, Dae-Jin Yun, Sang Yeol Lee, Keyan Zhu-Salzman,