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.

FIG 1

Structure of the catalytic domain of Cel7A with labeled glycosylation sites. The cellulose chain bound in the tunnel is shown as a cyan stick model. The cellobiose unit is shown as an orange stick model. The structure was simulated and displayed using PyMOL (DeLano Scientific [http://www.pymol.org]).

FIG 2

The absence of N-glycosylation of CBH1 hardly affects its extracellular secretion. (A) Extracellular protein concentration of strains expressing WT and mutant CBH1. (B) pNPC hydrolytic activity of the culture supernatant for strains expressing CBH1 variants after 60 and 80 h of induction with cellulose. The values in panels A and B are means of three biological replicates. Error bars show the standard deviations of the mean. (C) Western blot analysis of CBH1 secreted into the culture supernatant from mutant and WT strains after induction with 1% Avicel. Equal amount of culture supernatant relative to biomass was loaded for all strains.

FIG 3

Elimination of N-linked glycosylation in CBH1 induces expression of the UPR target genes. (A) Quantitative RT-PCR analysis of cbh1, bip1, pdi1, and cne1 gene expression in WT and CBH1 variant strains after induction with 1% (wt/vol) Avicel for different time periods. The values are means of three independent experiments. Error bars are the standard deviations of the mean. (B) Western blot analysis of CBH1 secreted into the culture supernatant from wild-type and Δcne1 strains expressing WT and MT CBH1, respectively, after induction with 1% Avicel. Equal amounts of culture supernatant relative to biomass were loaded for all strains.

FIG 4

Avicel hydrolysis by purified WT and mutant CBH1 proteins. (A) SDS-PAGE analysis of purified CBH1 variant proteins. Lanes 1 to 7 correspond to WT, M45, M270, M384, MD1, MD2, and MT CBH1, respectively. (B) Cellobiose production velocity curves of CBH1 variants. Avicel hydrolysis was carried out at 50°C and pH 5.0 with 1 μmol of purified WT and mutant CBH1 proteins. Duplicate assays were performed at each time point. Soluble products were determined by the DNS method using cellobiose as a standard as described in Materials and Methods. The velocities were calculated using origin7.5 (OriginLab, USA) and fitted to the Michaelis-Menten equation.

FIG 5

N-glycosylations at Asn384 and Asn45 are important for the thermal reactivity of CBH1. (A) pNPC hydrolytic activity of purified WT and MT CBH1 at different temperatures. (B) Thermal stability of CBH1 variants at 62°C. (C) Progress curves of pNPC hydrolysis by CBH1 variants at 62°C. (D) Product (cellobiose) velocity curves of CBH1 variants. Avicel hydrolysis was carried out as Fig. 4B except the reaction was allowed to occur at 62°C. (E) Hydrolytic activity of WT and MT CBH1 preincubated at 62°C for 25 min without Avicel. The preincubated proteins were then added to the reaction mixture with Avicel, and the cellobiose released was determined after 2 and 4 h of incubation.

FIG 6

Effect of temperature and pH on the secondary structure of CBH1 variants monitored by far-UV CD spectroscopy. (A) Spectral file of CBH1 variants at 25°C. (B) Spectral file of CBH1 variants preincubated at 62°C for 10 min. (C) CD spectra of WT and MT CBH1 at pH 4.8 and 8.0.