Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum

Abstract

Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. N-linked oligosaccharides are important for protein folding and stability, biosynthetic quality control, intracellular traffic and the physiological function of many Nglycosylated proteins. In metazoan organisms, the oligosaccharyltransferase is composed of a catalytic subunit (STT3A or STT3B) and a set of accessory subunits. Duplication of the catalytic subunit gene allowed cells to evolve OST complexes that act sequentially to maximize the glycosylation efficiency of the large number of proteins that are glycosylated in metazoan organisms. We will summarize recent progress in understanding the mechanism of (a) cotranslational glycosylation by the translocation channel associated STT3A complex, (b) the role of the STT3B complex in mediating cotranslational or posttranslocational glycosylation of acceptor sites that have been skipped by the STT3A complex, and (c) the role of the oxidoreductase MagT1 in STT3B-dependent glycosylation of cysteine-proximal acceptor sites.

The atomic structure of a eukaryotic oligosaccharyltransferase complex

“…Saccharomyces cerevisiae has two OST isoforms each with eight membrane proteins: the isoforms contain either Ost3 or Ost6 plus seven shared components: Ost1, 2, 4, and 5; Stt3; Wbp1; and Swp1 15 . All these subunits have homologs in the metazoan OST 2 : ribophorin I corresponds to the yeast Ost1, DAD1 to Ost2, N33/MagT1 or DC2/KCP2 to Ost3/6, OST4 to Ost4, TMEM258 to Ost5, OST48 to Wbp1, STT3A/STT3B to Stt3, and ribophorin II to Swp1 16 . Crystal structures of the Ost6 lumenal domain revealed a thioredoxin fold (TRX) 17,18 .…”

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-β-N-acetylglucosaminidase

“…Dolichol-linked oligosaccharides (DLOs) 3 are glycan donor substrates for asparagine (N)-linked glycosylation in mammals (1,2). The biosynthesis of DLOs starts on the cytosolic side of the endoplasmic reticulum (ER) membrane with the assembly of Man 5 GlcNAc 2 -PP-Dol (1).…”

“…This biosynthetic intermediate is flipped into the ER lumen and is converted to Glc 3 Man 9 GlcNAc 2 -PP-Dol. The fully assembled DLO is then transferred onto nascent polypeptides by the oligosaccharyltransferase (3).…”

Using glyco-engineering to produce therapeutic proteins

“…Consequently, glycosylation sites may be skipped leading to underglycosylation of recombinant proteins or additional sites may be used leading to aberrant glycosylation. Protein intrinsic factors like the surrounding amino-acid sequence and secondary structure, the positioning of the consensus sequence within the polypeptide as well as the presence of other protein modifications like disulfide bond formation contribute to N-glycosylation efficiency [4]. Recognition of these glycoprotein-specific features depends on the presence and function of the different OST subunits.…”