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Distinct roles of N- and O-glycans in cellulase activity and stability

Significance Glycosylation is a ubiquitous posttranslational modification of proteins wherein carbohydrates are appended to protein side chains, with myriad functions in molecular and cell biology. The enzymes that break down polysaccharides and other recalcitrant polymers in nature are often decorated with two canonical forms of glycosylation, N- and O-linked glycans, the roles of which are only partially understood in these key enzyme families with importance to both natural biomass turnover and industrial biotechnology. Here, we report that, depending on where they are attached, glycans play substantially different roles for the enzyme in thermal and proteolytic stability, substrate binding, and substrate turnover. Overall, these results provide fundamental insights into how glycans affect critical properties of biomass-degrading enzymes. In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)—a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall–degrading enzymes.