The expanding horizons of asparagine-linked glycosylation

Abstract

Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the past decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the determination of the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis among the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc(3)Man(9)GlcNAc(2)), heterogeneity is introduced into this glycan structure after it is transferred to the protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.

Figure 1

Pathway of N-linked glycosylation at the ER membrane in S. cerevisiae.

Figure 2

Structure of the eukaryotic N-linked glycan, Glc₃Man₉GlcNAc₂.

Figure 3

Comparison of predicted membrane topology of oligosaccharyltransferases from all three domains of life: (A) OT complex from S. cerevisiae; (B) PglB from C. jejuni; (C) AglB from M. voltae. The size of the membrane loops between the transmembrane domains in (B) and (C) is variable and has not yet been determined.

Figure 4

The calnexin/calreticulin pathway of quality control for protein folding. (1) Nascent proteins travel through the translocon and are glycosylated by OT; (2) The two terminal glucose residues are removed by glucosidase I and II; (3) The folding protein interacts with the chaperones calnexin and calreticulin and the ERp57 oxidoreductase. If misfolded, they are recognized by UGGT and cycled through the pathway again; (4) Defective proteins are demannosylated and ultimately sent for degradation through the ERAD pathway, while properly folded proteins exit the cycle.

Figure 5

Structure of the N-linked GlcGalNAc₅Bac heptasaccharide identified in C. jejuni.

Figure 6

N-linked glycosylation pathway in C. jejuni.

Figure 7

Structures of representative archaeal N-linked glycans. (A) H. halobium, shorter glycan; (B) H. halobium, longer glycan; (C) M. fervidus, in which the two residues at the non-reducing end are either Man or Glc; (D) S. acidocaldarius, containing the unique 6-sulfoquinovose moiety; (E) M. voltae, where the R indicates the presence of an unidentified hexose in some strains; (F) M. maripaludis; (G) T. acidophilum, in which the order of the residues near the reducing end are unclear. Unusual sugar moieties are colored in blue; see text for more detail and specific references.

Figure 8

Current models of N-linked glycosylation in (A) M. voltae; (B) M. maripaludis; and (C) H. volcanii based on recent genetic studies. The exact structure of the H. volcanii N-linked glycan has not yet been determined, but data suggest that it is a pentasaccharide as indicated.