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

Abstract

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.

The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation

“…Another issue concerns the effect of glycosylation on linker structure and dynamics. Such glycosylation is known to happen in CAZymes from fungi (39,45) and actinomycetes (46), and its impact is currently receiving considerable attention (39,45). In their study on the C. fimi xylanase, Poon et al (43) concluded that glycosylation of the 20-residue Pro-Thr linker had limited effects on linker structure and dynamics.…”

“…Studies on the roles of glycosylated linkers in fungal modular CAZymes have revealed an impact of glycosylation on substrate binding and proteolytic resistance, but information on the impact of glycosylation on linker shape and dynamics is scarce. Interestingly, in a recent study, Amore et al (45) concluded that glycosylation of the linker in a fungal cellobiohydrolase ensures the separation between the catalytic domain and the CBM. The present data show that the nonglycosylated linker of ScLPMO10C has an extended conformation that separates the domains.…”

Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

“…C) and in its close homolog Th Cel7B . Although it is widely acknowledged that a combination of several factors are responsible for stabilizing thermophilic structures , we note that the features listed above point as elements contributing to the high T m of Re Cel7B . In addition to this, the presence of N‐glycans is probably important for the very high solubility of Re Cel7B, another desirable trait from an industrial perspective .…”

Systematic modification of N-glycosylation sites in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

“…Due to the complexity, recalcitrance and insolubility of the plant biomass [2], high enzyme titers must be used to ensure e cient biomass hydrolysis, and this challenges the economic feasibility of the process. To overcome this, extensive research has sought to either engineer catalytically more e cient enzymes or to develop more e cient expression hosts such as Trichoderma reesei [3], Saccharomyces cerevisiae [4] and Aspergillus niger [5]. The latter effort has enabled industrial production of cellulases, but usually with a range of isoforms with different apparent molecular weights [6].…”

“…Most of the N-glycans on TrCel7A have been characterized as high-mannose type (Man), containing from Man 5-9 residues linked to a chitobiose core of two N-acetylglucosamine units (GlcNAc) 2 , whereas the O-glycans consist mainly of Man 1-4 randomly distributed in both the linker region and CBM domain with the majority bound to the linker of TrCel7A [7,10]. Recently, Amore et al (2017) [3] performed extensive mass spectrometry (MS) characterization of the different N-glycoforms of TrCel7A expressed in T. reesei, and this work indicated a broader complexity of N-glycans, including the presence of fucose, galactose or additional Nacetylglucosamine residues. The glycan complexity is not only in uenced by the expression hosts and their extracellular activities of glycosidases and transferases, but also the composition of the growth media [11].…”