N-linked protein glycosylation in the ER

Abstract

N-linked protein glycosylation in the endoplasmic reticulum (ER) is a conserved two phase process in eukaryotic cells. It involves the assembly of an oligosaccharide on a lipid carrier, dolichylpyrophosphate and the transfer of the oligosaccharide to selected asparagine residues of polypeptides that have entered the lumen of the ER. The assembly of the oligosaccharide (LLO) takes place at the ER membrane and requires the activity of several specific glycosyltransferases. The biosynthesis of the LLO initiates at the cytoplasmic side of the ER membrane and terminates in the lumen where oligosaccharyltransferase (OST) selects N-X-S/T sequons of polypeptide and generates the N-glycosidic linkage between the side chain amide of asparagine and the oligosaccharide. The N-glycosylation pathway in the ER modifies a multitude of proteins at one or more asparagine residues with a unique carbohydrate structure that is used as a signalling molecule in their folding pathway. In a later stage of glycoprotein processing, the same systemic modification is used in the Golgi compartment, but in this process, remodelling of the N-linked glycans in a protein-, cell-type and species specific manner generates the high structural diversity of N-linked glycans observed in eukaryotic organisms. This article summarizes the current knowledge of the N-glycosylation pathway in the ER that results in the covalent attachment of an oligosaccharide to asparagine residues of polypeptide chains and focuses on the model organism Saccharomyces cerevisiae. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

“…On the other hand, core α1,3-fucosylation is abolished and processing by mannosidases and the bisecting N-acetylglucosaminyltransferase is strongly reduced. The previous data on wild-type Hex 8 HexNAc 3 (PMe) 2 (43) and our own unpublished data regarding the major wild-type anionic N-glycans (Hex 8 HexNAc 2 (PMe) 2 and Hex 8 HexNAc 3 PMeS 2 ) indicate the presence of methylphosphate on the A and C branches of a Man8A scaffold. The surprise in the mutant is with the anionic N-glycans; in this case the most dominant structure is Hex 9 HexNAc 3 PMe, with methylphosphate on the B or C branches, but which lacks any glucose residues Therefore the question arises as to how the glucose residues are removed before the glycoproteins reach their final molecular form in the mutant.…”

“…Hex 9-10 HexNAc 2 (PMe) 2 , resulted in a fragment of m/z 997 (Hex 5 [PMe] 2 ) suggesting that both the B and C branches were modified with terminal methylphosphate residues ( Figure 6G). An alternative form of di-anionic glycan species is represented by Hex 8-10 HexNAc 2-3 PMeS structures; MS/MS showed the presence of m/z 255 (Hex 1 PMe) and 403 (Hex 2 S) and, in some cases, 497 (Hex 2 PMeS) fragments ( Figure 6E and H).…”

“…However, a high variability in terms of finally-processed structures is obvious when comparing different species, which is reflected also in the types of enzymes present. Nevertheless, amongst eukaryotes which are not unicellular obligate parasites, the initial stages of the N-glycosylation process (precursor formation, oligosaccharyl transfer and initial trimming) are highly conserved (2) . After transfer of a tetradecasaccharide containing three glucose, nine mannose and two N-acetylglucosamine residues (Glc 3 Man 9 GlcNAc 2 ) to protein, the three glucose residues are removed by α-glucosidases I and II, which are resident in the endoplasmic reticulum and have a neutral pH optimum (3,4) .…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…The N-linked glycosylation of proteins in the ER is an important prerequisite for protein targeting (Hubbard and Ivatt 1981;Aebi, 2013). Nearly all eukaryotic OSTs use the membrane-bound dolichol pyrophosphate GlcNAc 2 Man 9 Glc 3 as substrate (Kelleher et al, 2003).…”

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

“…Yeast OST is composed of eight subunits (Ost1, Ost2, Ost3/Ost6, Ost4, Ost5, Stt3, Swp1, and Wbp1), which are assembled in the ER membrane in large heterooligomeric complexes alternatively containing Ost3 or Ost6 (reviewed in Ref. 6). It is well established that N-glycosylation can be temporally coupled to the protein translocation reaction to ensure the accessibility of glycosylation sites before the acceptor protein starts to fold, and direct interactions between OST and the TC have been demonstrated (reviewed in Ref.…”