Kolaczkowski_2021_Glycobiology__

Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce non-native glycan chains which may not fulfil the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exo-glycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 alpha-1,2-mannosidase from Neobacillus novalis, a GH2 beta-galactofuranosidase from Amesia atrobrunnea and the jack bean alpha-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.