Analysis of fungal high-mannose structures using CAZymes

Bartłomiej M. Kołaczkowski, Christian I. Jørgensen, Nikolaj Spodsberg, Mary A. Stringer, Nitin T. Supekar, Parastoo Azadi, Peter Westh, Kristian B.R.M. Krogh, Kenneth Jensen*

*Corresponding author

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review


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 nonnative glycan chains that may not fulfill 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 exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 β-galactofuranosidase from Amesia atrobrunnea and the jack bean α-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.

Udgave nummer4
Sider (fra-til)304-313
Antal sider10
StatusUdgivet - 1 apr. 2022

Bibliografisk note

Funding Information:
Roskilde University, Novozymes A/S, Innovation Fund Denmark [Grant number: 5150-00020B], the Novo Nordisk Foundation [Grant number: NNF15OC0016606 and NNFSA170028392] and the Carlsberg Foundation.


  • Aspergillus oryzae
  • cellobiohydrolase
  • fungal glycoproteins
  • glycoside hydrolase
  • O-glycosylation

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