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PreprintCited by 17Glycosylation and Glycoproteins ResearchCarbohydrate Chemistry and SynthesisViral Infectious Diseases and Gene Expression in Insects

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Global view of domain-specific O-linked mannose glycosylation in glycoengineered cells

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Povolo, Lorenzo, Tian, Weihua, Vakhrushev, Sergey Y., Halim, Adnan·2024·Molecular & Cellular Proteomics
DOI 10.1101/2024.01.15.575371PMC PMC11292533

Preprint — published as 10.1016/j.mcpro.2024.100796 · Paper page

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Abstract

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved post-translational modification (PTM) that fulfills important biological roles during embryonic development. Three non-redundant enzyme families, POMT1/POMT2, TMTC1-4 and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knock-out (KO) of O-Man glycosyltransferase genes. We established a human cell library for analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man glycosylated adhesion molecules and receptors.

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Metadata

Authors
Povolo, L., Tian, W., Vakhrushev, S. Y., & Halim, A.
Affiliations
University of Copenhagen (DK)
Corresponding author
Halim, Adnan
ORCID
Venue
Molecular & Cellular Proteomics
Publisher
openRxiv
Published
2024-01-16
Volume
23
Issue
7
Pages
100796-100796
ISSN
1535-9476 (ISSN-L)
DOI
10.1101/2024.01.15.575371
PMC
PMC11292533
OpenAlex
W4399391731
Cited by
17
References
1
Citations by year
2026: 12 · 2025: 3 · 2024: 2
FWCI
2.31
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Molecular & Cellular Proteomics (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
Funders
  • Villum Fonden (00025438)
  • Mizutani Foundation for Glycoscience
  • National Research Foundation
  • Danmarks Grundforskningsfond (DNRF‐107)
  • National Institute of Diabetes and Digestive and Kidney Diseases (K24DK002800)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Keywords
glycoproteomics, glycosyltransferase substrate specificity, immunoglobulin-like domains, plasma membrane proteins, human cell lines, adhesion molecules, extracellular matrix interactions
MeSH terms
Cell Line, Glycoproteins, Glycosylation, Humans, Mannose, Protein Processing, Post-Translational, Substrate Specificity, Glycosyltransferases, Proteomics, Cell Engineering

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