Analysis of the role of O‐glycosylation in GH51 α‐l‐arabinofuranosidase from Pleurotus ostreatus

Abstract

In this study, the recombinant α‐l‐arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site‐directed mutagenesis with the aim of elucidating the role of glycosylation on the properties of the enzyme at the level of S160 residue. As a matter of fact, previous mass spectral analyses had led to the localization of a single O‐glycosylation at this site. Recombinant expression and characterization of the rPoAbf mutant S160G was therefore performed. It was shown that the catalytic properties are slightly changed by the mutation, with a more evident modification of the K cat and K M toward the synthetic substrate pN‐glucopyranoside. More importantly, the mutation negatively affected the stability of the enzyme at various pHs and temperatures. Circular dichroism (CD) analyses showed a minimum at 210 nm for wild‐type (wt) rPoAbf, typical of the beta‐sheets structure, whereas this minimum is shifted for rPoAbf S160G, suggesting the presence of an unfolded structure. A similar behavior was revealed when wt rPoAbf was enzymatically deglycosylated. CD structural analyses of both the site‐directed mutant and the enzymatically deglycosylated wild‐type enzyme indicate a role of the glycosylation at the S160 residue in rPoAbf secondary structure stability.

Molecular-scale features that govern the effects of O-glycosylation on a carbohydrate-binding module

“…A well-established effect of protein glycosylation is an increase in proteolytic stability, either by increasing the rigidity of the protein, or by providing a steric barrier that hinders protease access to the peptide bonds. 19 , 26 – 28 Our results indicate that steric hindrance may be less important than peptide rigidity in the case of CBM O -glycosylation. Support for this conclusion comes from the CBM variants 4–19 ( Fig.…”

The impact ofO-glycan chemistry on the stability of intrinsically disordered proteins

“…Analyses are reported on the T-REMD population from the lowest temperature replica (300 K). Two hypotheses for the increased proteolytic stability imparted by glycans are that (i) glycans increase protein rigidity 20 , 21 and that (ii) glycans impart steric hindrance to restrict protease access. 22 Both hypotheses were tested computationally by examining differences in protein flexibility and accessibility.…”

Protein Glycosylation Investigated by Mass Spectrometry: An Overview

“…The intense O-mannosylation characterized the O-glycosylated proteins displaying a higher variable in sugar components and the linkage type of glycans determining the multiple functions [27]. For instance, the catalytic activity of α-L-arabinofuranosidase of fungus Pleurotus ostreatus relied in O-glycosylation of S160 residue crucial for enzyme structural stability [28].…”

“…Other authors lingered on the spike (S) envelope protein of the currently emerging virus (CoV) inducing severe acute respiratory syndrome (SARS) to explain the crucial role of glycoprotein in infection initiation by binding receptor-binding domain of S protein to the cellular receptor ACE2 and in the phase of viral envelope fusion with the host-cell membrane through the endosomal pathway [44]. Actually, the S proteins of coronaviruses display a larger number of N-linked glycan sites (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) per protomer but a lower population of oligomannose-type glycans (30%) compared to the other viruses [45]. The increase of the number of glycosylation sites and the reduced density by oligomannose-type glycans seems to be based on an evolutive selection, reflecting a balance between immune evasion by epitope shielding and functionality by attachment to the host cell.…”