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.
Department of Entomology, Texas A&M University, College Station, 77843, USA.
Yong Hun
YH
Chi
Yoon Duck
YD
Koo
Susie Y
SY
Dai
Ji-Eun
JE
Ahn
Dae-Jin
DJ
Yun
Sang Yeol
SY
Lee
Keyan
K
Zhu-Salzman
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