Distinct Roles of N- and O-Glycans in Cellulase Activity and Stability

Gregg Beckham, Brandon Knott, Jeffrey Linger, Todd VanderWall, John Yarbrough, Michael Crowley, Michael Himmel, Steve Decker, Sarah Hobdey, Nitin Supekar, Asif Shajahan, Parastoo Azadi, Peng Zhao, Lance Wells, Zhongping Tan, Antonella Amore, Todd Shollenberger, Larry Taylor

Research output: Contribution to journalArticlepeer-review

74 Scopus Citations


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 biomassdegrading 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 thewhole-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.

Original languageAmerican English
Pages (from-to)13667-13672
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number52
StatePublished - 26 Dec 2017

NREL Publication Number

  • NREL/JA-5100-70178


  • Cellulase
  • Glycoside hydrolase
  • Glycosylation
  • Intrinsically disordered protein
  • Mannosylation


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