Molecular Determinants of Sequence-Dependent N-glycosylation by the Human Oligosaccharyltransferase
- 1Universitat de Valencia
Abstract
Protein N-glycosylation is catalysed by the oligosaccharyltransferase (OST) complex, yet how local sequon composition controls substrate selection by human OST remains poorly understood. Here we combine atomistic molecular dynamics, alchemical free-energy calculations, in cellulo glycosylation assays and human glycoproteome analysis to define the molecular basis of sequence-dependent N-glycosylation. We built, to our knowledge, the first atomistic model of human OST with both the lipid-linked oligosaccharide donor and an acceptor peptide simultaneously accommodated in a catalytically competent complex. Simulations show that conserved STT3 motifs stabilize the acceptor Asn and explain how substitutions at positions 0, +1 and +2 alter binding and productive geometry. Cellular assays in HEK 293T cells validate the predicted loss of glycosylation for N0Q sequon, +1 Pro and the +2 hierarchy Thr > Ser >> Cys. Analysis of 14,800 human glycosites confirms these rules at proteome scale.
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BibTeX
@misc{andjelkovic2026molecular,
title = {{Molecular Determinants of Sequence-Dependent N-glycosylation by the Human Oligosaccharyltransferase}},
author = {M. Andjelkovic and J. F. Belen-Aguilar and D. Belda and J. J. Ruiz-Pernia and I. Tunon and I. Mingarro},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.03.749169},
url = {https://www.biorxiv.org/content/10.64898/2026.09.03.749169v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Molecular Determinants of Sequence-Dependent N-glycosylation by the Human Oligosaccharyltransferase
AU - M. Andjelkovic
AU - J. F. Belen-Aguilar
AU - D. Belda
AU - J. J. Ruiz-Pernia
AU - I. Tunon
AU - I. Mingarro
PY - 2026
DA - 2026/09/03
PB - bioRxiv
DO - 10.64898/2026.09.03.749169
UR - https://www.biorxiv.org/content/10.64898/2026.09.03.749169v1
AB - Protein N-glycosylation is catalysed by the oligosaccharyltransferase (OST) complex, yet how local sequon composition controls substrate selection by human OST remains poorly understood. Here we combine atomistic molecular dynamics, alchemical free-energy calculations, in cellulo glycosylation assays and human glycoproteome analysis to define the molecular basis of sequence-dependent N-glycosylation. We built, to our knowledge, the first atomistic model of human OST with both the lipid-linked oligosaccharide donor and an acceptor peptide simultaneously accommodated in a catalytically competent complex. Simulations show that conserved STT3 motifs stabilize the acceptor Asn and explain how substitutions at positions 0, +1 and +2 alter binding and productive geometry. Cellular assays in HEK 293T cells validate the predicted loss of glycosylation for N0Q sequon, +1 Pro and the +2 hierarchy Thr > Ser >> Cys. Analysis of 14,800 human glycosites confirms these rules at proteome scale.
ER -