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 for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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 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.
Original languageEnglish
Article numbercwab127
JournalGlycobiology
Volume32
Issue number4
Pages (from-to)304-313
Number of pages10
ISSN0959-6658
DOIs
Publication statusPublished - 2022

Keywords

  • Aspergillus oryzae
  • Cellobiohydrolase
  • Fungal glycoproteins
  • Glycoside hydrolase
  • O-glycosylation

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