Glycobiology Advance Access originally published online on December 23, 2003
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Glycobiology vol 14 no 5 pp. 417-429, 2004
Glycobiology vol. 14 no. 5 © Oxford University Press 2004; all rights reserved.
Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris
2 Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1; 3 Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8; 4 Department of Agricultural Science, Korea National Open University,169, Dongsung-Dong, Chongro-Ku, Seoul, 110-791, Korea
Received on August 1, 2003; revised on October 16, 2003; accepted on October 21, 2003
A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar Km values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.
1 To whom correspondence should be addressed; e-mail: ryada{at}uoguelph.ca