Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris.
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 K(m) 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.
Yoshimasu
Mark A
MA
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Tanaka
Takuji
T
Ahn
Jong-Kun
JK
Yada
Rickey Y
RY
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000235:genetics*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
D017931:DNA Primers; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A