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

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 > or =50kDa 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.

published proceedings

  • Comp Biochem Physiol B Biochem Mol Biol

author list (cited authors)

  • Chi, Y. H., Koo, Y. D., Dai, S. Y., Ahn, J., Yun, D., Lee, S. Y., & Zhu-Salzman, K.

citation count

  • 11

complete list of authors

  • Chi, Yong Hun||Koo, Yoon Duck||Dai, Susie Y||Ahn, Ji-Eun||Yun, Dae-Jin||Lee, Sang Yeol||Zhu-Salzman, Keyan

publication date

  • May 2010