title: 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.
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People's Republic of China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Qi
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People's Republic of China.
authors.forename: Weixin
authors.initials: W
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People's Republic of China.
authors.forename: Fengjie
authors.initials: F
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People's Republic of China.
authors.forename: Guanjun
authors.initials: G
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People's Republic of China weifliu@sdu.edu.cn.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Liu
mesh_terms: D020134:Catalytic Domain; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D006031:Glycosylation; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D043366:Cellulose 1,4-beta-Cellobiosidase
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