Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells
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Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells. / Povolo, Lorenzo; Tian, Weihua; Vakhrushev, Sergey Y.; Halim, Adnan.
In: Molecular & cellular proteomics : MCP, Vol. 23, No. 7, 100796, 2024.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells
AU - Povolo, Lorenzo
AU - Tian, Weihua
AU - Vakhrushev, Sergey Y.
AU - Halim, Adnan
N1 - Publisher Copyright: Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.
PY - 2024
Y1 - 2024
N2 - Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant 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 knockout of O-Man glycosyltransferase genes. We established a human cell library for the 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.
AB - Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant 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 knockout of O-Man glycosyltransferase genes. We established a human cell library for the 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.
KW - cadherin
KW - CRISPR/cas
KW - dystroglycan
KW - genetic engineering
KW - glycobiology
KW - lectins
KW - mass spectrometry (MS)
KW - O-man
KW - plexin
KW - post-translational modification (PTM)
KW - protein domains
KW - protein O-mannosyltransferases
U2 - 10.1016/j.mcpro.2024.100796
DO - 10.1016/j.mcpro.2024.100796
M3 - Journal article
C2 - 38851451
AN - SCOPUS:85199933747
VL - 23
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 7
M1 - 100796
ER -
ID: 402284385