Understanding the role of O- and N-glycosylation on the structure and activity of Trichoderma reesei Cel7A

Fungal cellulases are now being utilized industrially to depolymerize cellulose in ethanol biorefineries. In particular, cellobiohydrolases from Glycoside Hydrolase Family 7 (GH7) are the primary workhorse enzymes in these cocktails.  Typically,  these enzymes are multi-modular with a large catalytic domain and samll Family 1 carbohydrate-binding module connected by a flexible linker peptide. When expressed in fungal hosts, GH7 cellobiohydrolases exhibit N- and O-linked glycosylation, with the former present on the catalytic domain and the latter on all three sub-domains. Glycosylation on these enzymes is thought to primarily play a role in proteolysis protection in nature, but previous studies have also demonstrated that the type and extent of glycosylation has a dramatic effect on activity. Indeed, many questions remain as to the role of both N- and O-linked glycans regarding their impact on activity, structure, and thermal and proteolytic stability.

To deepen our collective understanding of the role of glycosylation on the archetypal GH7 cellulase, namely Cel7A from Trichoderma reesei, a systematic investigation of N- and O-linked glycans have been performed via mutational analyses. Multiple mutants alternatively lacking one or more N-glycosylation sites of the catalytic domain, the O-glycosylation sites of the carbohydrate-binding module, and the O-glycosylation sites of the linker peptide were designed and expressed in a T. reesei constitutive expression system (Linger et al., 2015). The O-glycan and N-glycan mutants have been characterized for enzyme activity and stability, and the results demonstrate the myriad ways that glycosylation influences critical properties of Cel7A for industrial performance.