The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 function as ubiquitous and selective modulators of mammalian N-glycosylation

Abstract

SummaryProtein N-glycosylation is an essential modification that occurs in all eukaryotes and is catalysed by the oligosaccharyltransferase (OST) in the endoplasmic reticulum. Comparative studies have clearly shown that eukaryotic STT3 proteins alone can fulfil the enzymatic requirements for N-glycosylation, yet in many cases STT3 homologues form stable complexes with a variety of non-catalytic OST subunits. Whereas some of these additional components might play a structural role, others appear to increase or modulate Nglycosylation efficiency for certain precursors. Here, we have analysed the roles of three non-catalytic mammalian OST components by studying the consequences of subunit-specific knockdowns on the stability and enzymatic activity of the OST complex. Our results demonstrate that OST48 and DAD1 are required for the assembly of both STT3A-and STT3B-containing OST complexes. The structural perturbations of these complexes we observe in OST48-and DAD1-depleted cells underlie their pronounced hypoglycosylation phenotypes. Thus, OST48 and DAD1 are global modulators of OST stability and hence N-glycosylation. We show that KCP2 also influences protein N-glycosylation, yet in this case, the effect of its depletion is substrate specific, and is characterised by the accumulation of a novel STT3A-containing OST subcomplex. Our results suggest that KCP2 acts to selectively enhance the OST-dependent processing of specific protein precursors, most likely co-translational substrates of STT3A-containing complexes, highlighting the potential for increased complexity of OST subunit composition in higher eukaryotes.

N-linked protein glycosylation in the ER

“…In contrast, lumen-exposed domains of larger subunits can directly interact with polypeptide substrates and prevent their folding before glycosylation has taken place. Depletion of the mammalian ribophorin I and KCP2 subunit affected the glycosylation of only a subset of proteins [109][110][111] compatible with their role as substrate-specific chaperone. An enzymatically active role of one of the OST subunits has been demonstrated for the yeast Ost6p: the luminal domain of this protein forms has a thioredoxin fold and can function as an oxidoreductase in vitro [112].…”

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

“…In addition, ribophorin I interacts with a subset of polypeptide substrates and presents them to the catalytic subunit STT3 to facilitate efficient glycosylation of these proteins (Wilson and High 2007;Wilson et al 2008). Knockdown of OST48 and DAD1 by small interfering RNA (siRNA) results in destabilization of both OST complex isoforms (Roboti and High 2012b). KCP2 associates mainly, or even exclusively, with STT3A complex isoforms, and knockdown experiments led to the conclusion that KCP2 may facilitate the glycosylation of selected substrate proteins (Roboti and High 2012a,b).…”

Glycosylation of closely spaced acceptor sites in human glycoproteins

“…Depletion of non-catalytic OST subunits (ribophorin I, OST48 or DAD1) that are shared by both the STT3A and STT3B complexes generally cause a global defect in N-linked glycosylation because accessory subunit loss reduces the stability of both the STT3A and STT3B complexes (Roboti and High, 2012;Ruiz-Canada et al, 2009;Wilson and High, 2007). Likewise, fibroblasts from CDG patients with a mutation in the DDOST gene, which encodes OST48, have a general defect in Nlinked glycosylation (Jones et al, 2012).…”