Molecular Determinants of Sequence-Dependent N-glycosylation by the Human Oligosaccharyltransferase
Year: 2026
Type: preprint
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.
Institutions: University of Bayreuth, Universitat de València
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G1175115578, G1352746561, G1422035950, G1622782223, G1927686253, G2262748287, G300979063, G3220251042, G34346518, G3480869486, G3565166246, G3565621276, G3857581125, G410571670, G425668148, G451917667, G4526231891, G464105488, G5080475149, G5539308778, G5967599077, G5987682017, G6225180989, G700394663, G7084143925, G792975244, G929308097
OpenAlex ID: W7211911822