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

作者：Chi Yong Hun; Koo Yoon Duck; Dai Susie Y; Ahn Ji Eun; Yun Dae Jin; Lee Sang Yeol; Zhu Salzman Keyan^*

来源：Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 2010, 156(1): 40-47.

DOI：10.1016/j.cbpb.2010.01.017

• Cathepsin B
• Counter-defense
• N-glycosylation
• Pichia
• Site-directed mutagenesis

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 (Asn(100) and Asn(236)) in CmCatB. Asn to Gln substitution at Asn(100)-X-Cys on the background of the double mutation at the canonical sites (m1m2. Asn(97)-> Gln and Asn(207)-> Gln) resulted in a single discrete band on the gel, namely m1m2c1 (Asn(97)-> Gln, Asn(207)-> Gln and Asn(100)-> Gln). However, another triple mutant protein m1m2c2 (Asn(97)-> Gln, Asn(207)-> Gln and Asn(236)-> Gln) and quadruple mutant protein m1m2c1c2 were unable to be expressed in Pichia cells. Thus Asn(236) appears necessary for protein expression while Asn(100) is responsible for non-canonical glycosylation. Removal of carbohydrate moieties, particularly at Asn(100), substantially enhanced proteolytic activity but compromised protein stability. Thus, glycosylation could significantly impact biochemical properties of CmCatB.

• 出版日期2010-5