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

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• 作者：Yong Hun Chi ; Yoon Duck Koo ; Susie Y. Dai ; Ji-Eun Ahn ; Dae-Jin Yun ; Sang Yeol Lee ; Keyan Zhu-Salzman
• 关键词：Cathepsin B ; Counter-defense ; N-glycosylation ; Pichia ; Site-directed mutagenesis
• 刊名：Comparative Biochemistry and Physiology, Part B
• 出版年：2010
• 出版时间：May 2010
• 年：2010
• 卷：156
• 期：1
• 页码：40-47
• 全文大小：1396 K

文摘

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.