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 > 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.
Chi
Yong Hun
YH
Department of Entomology, Texas A&M University, College Station, 77843, USA.
Koo
Yoon Duck
YD
Dai
Susie Y
SY
Ahn
Ji-Eun
JE
Yun
Dae-Jin
DJ
Lee
Sang Yeol
SY
Zhu-Salzman
Keyan
K
D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D002401:Cathepsin B / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
D019476:Insect Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D006026:Glycoside Hydrolases; D002401:Cathepsin B; D003545:Cysteine