Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris

Abstract

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.

Expression, purification and characterization of a phyA m -phyCs fusion phytase

“…After deglycosylation by EndoH f , the phyA m , phyCs and fusion phyA m -phyCs phytases had an apparent molecular size of 51, 42 and 95 kDa, respectively. The glycosylation may have a number of effects on the properties of an enzyme, and it may have a positive effect on the thermostability (Yoshimasu et al, 2004;). As we previously reported, the glycosylation of P. pastoris phytase appeared to have increased with induction time and the glycosylated phytase resulted in the enhancement of thermostability (Zou et al, 2006), so we deduced that deglycosylation may lead to the decrease of fusion phytase thermostability.…”

Pepsinogen C expression, regulation and its relationship with cancer

“…The PGC secondary structure is 15% α-helix and 44% β-sheet [1], illustrating PGC is β-sheet protein–rich. The PGC protein structure achieves stability through numerous hydrogen bonds; in addition, its N -glycosylation sites also minimise protein conformational alteration to stabilise the protein structure [11]. X-ray crystallography data had shown that the PGC protein tertiary structure was similar to the aspartic protease family and that it contained a plurality of aspartic protease family symbols and two rhodopsin-like G protein–coupled receptor symbols.…”

Cadherin flexibility provides a key difference between desmosomes and adherens junctions

“…Glycosylation is an important modulator of rigidity in proteins (25)(26)(27), and type 1 cadherins are extensively glycosylated, with up to 9 N-and 16 O-glycosylated sites predicted (28,29), and 15 observed in cadherin crystal structures (3,24). In contrast, desmogleins have fewer predicted N-linked glycosylation sites than classical cadherins.…”