N-linked protein glycosylation in the endoplasmic reticulum

Abstract

The attachment of glycans to asparagine residues of proteins is an abundant and highly conserved essential modification in eukaryotes. The N-glycosylation process includes two principal phases: the assembly of a lipid-linked oligosaccharide (LLO) and the transfer of the oligosaccharide to selected asparagine residues of polypeptide chains. Biosynthesis of the LLO takes place at both sides of the endoplasmic reticulum (ER) membrane and it involves a series of specific glycosyltransferases that catalyze the assembly of the branched oligosaccharide in a highly defined way. Oligosaccharyltransferase (OST) selects the Asn-X-Ser/Thr consensus sequence on polypeptide chains and generates the N-glycosidic linkage between the side-chain amide of asparagine and the oligosaccharide. This ER-localized pathway results in a systemic modification of the proteome, the basis for the Golgi-catalyzed modification of the N-linked glycans, generating the large diversity of N-glycoproteome in eukaryotic cells. This article focuses on the processes in the ER. Based on the highly conserved nature of this pathway we concentrate on the mechanisms in the eukaryotic model organism Saccharomyces cerevisiae.

Figure 1.

Structure of the N-glycan. The N-glycan structure consists of 14 carbohydrate residues. It contains two N-acetylglucosamines (GlcNAc, black squares), nine mannoses (Man, gray circles), and three glucoses (Glc, black circles). Linkage information is indicated between carbohydrate residues. The different residues are labeled with the genes encoding the glycosyltransferase that catalyzes the transfer of the respective residue. The structure of the glycan has three branches labeled a, b, and c.

Figure 2.

The asparagine-linked glycosylation pathway. The biosynthesis of lipid-linked oligosaccharide (LLO) is catalyzed by a series of glycosyltransferases encoded by asparagine-linked glycosylation (ALG) genes. The buildup of the glycan is initiated at the cytoplasmic leaflet of the ER membrane by the addition of N-acetylglucosamine (GlcNAc)-phosphate to dolichol-phosphate (Dol-P) from nucleotide-activated UDP-GlcNAc. After the addition of a second GlcNAc residue to Dol-PP-GlcNAc, five mannoses (Man) are attached from GDP-Man to the LLO. After translocation into the ER lumen, four additional Man from Dol-P-Man and three glucoses are added from Dol-P-Glc. The oligosaccharide is then transferred en bloc to asparagine side chains of nascent polypeptides by the oligosaccharyltransferase (OST). The synthesis of dolichol and dolichol-bound monosaccharide precursors as well as proposed models for dolichol recycling are discussed in detail in the text.