Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei

Abstract

N-linked glycosylation modulates and diversifies the structures and functions of the eukaryotic proteome through both intrinsic and extrinsic effects on proteins. We investigated the significance of the three N-linked glycans on the catalytic domain of cellobiohydrolase I (CBH1) from the filamentous fungus Trichoderma reesei in its secretion and activity. While the removal of one or two N-glycosylation sites hardly affected the extracellular secretion of CBH1, eliminating all of the glycosylation sites did induce expression of the unfolded protein response (UPR) target genes, and secretion of this CBH1 variant was severely compromised in a calnexin gene deletion strain. Further characterization of the purified CBH1 variants showed that, compared to Asn270, the thermal reactivity of CBH1 was significantly decreased by removal of either Asn45 or Asn384 glycosylation site during the catalyzed hydrolysis of soluble substrate. Combinatorial loss of these two N-linked glycans further exacerbated the temperature-dependent inactivation. In contrast, this thermal labile property was less severe when hydrolyzing insoluble cellulose. Analysis of the structural integrity of CBH1 variants revealed that removal of N-glycosylation at Asn384 had a more pronounced effect on the integrity of regular secondary structure compared to the loss of Asn45 or Asn270. These data implicate differential roles of N-glycosylation modifications in contributing to the stability of specific functional regions of CBH1 and highlight the potential of improving the thermostability of CBH1 by tuning proper interactions between glycans and functional residues.

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance

“…Szengyel and Zacchi [27] showed that acetic acid had no inhibition effect on Trichoderma reesei cellulase activities, but increasing acetic acid concentrations improved BGL1 activity. Similar results on cellulases were reported by Qi et al [28], except that acetic acid had an inhibitory effect on BGL1. Formic acid is also known as a potent inhibitor of hydrolysis and has been shown to decrease glucose production, while increasing concentration of furfural inhibited both cellulase and BGL1 activities [29].…”

The consensus Nglyco-X-S/T motif and a previously unknown Nglyco-N -linked glycosylation are necessary for growth and pathogenicity ofPhytophthora

“…6). Similarly, previous work revealed that in Trichoderma reesei, abolishing N-glycosylation of CBH1 led to the induction UPR target gene expression, with 5-, 4-, and 2-fold up-regulation of CNE1, BIP1, and PDI1, respectively (72). These results thus demonstrated that elimination of glycosylation sites can lead to a range of different effects on the folding and stabilization of glycoproteins in oomycetes, though further exploration is needed to characterize the full extent and underlying mechanisms of these effects.…”

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

“…His tag fusion proteins were then expressed in E. coli BL21(DE3). The crude enzyme solutions were purified using a fast protein liquid chromatography system consisting of nickel-nitrilotriacetic acid (Ni-NTA) and DEAE columns (33).…”

“…Thus, N-glycosylation represents a stabilizing factor against proteolytic cleavage by proteases (29). Apart from conferring an increased resistance to proteolysis, N-glycosylation can also enhance enzyme thermostability and alter catalytic activity (30)(31)(32)(33). The improved conformational stability of enzymes derived from the steric interactions between N-linked glycan and protein can decrease the enzyme flexibility or increase the rigidity of the enzyme structure (34,35).…”