title: The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 function as ubiquitous and selective modulators of mammalian N-glycosylation.
abstract: Protein 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 N-glycosylation 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.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, UK.
authors.forename: Peristera
authors.initials: P
authors.lastname: Roboti
authors.forename: Stephen
authors.initials: S
authors.lastname: High
mesh_terms: D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D006031:Glycosylation; D006367:HeLa Cells; D056945:Hep G2 Cells; D006602:Hexosyltransferases / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D021122:Protein Subunits
chemical_list: D008565:Membrane Proteins; D021122:Protein Subunits; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase