N-linked protein glycosylation involves the transfer of an oligosaccharide precursor to the amide nitrogen atom of asparagine residues in the recognition sequence Asn-X-Ser/Thr, where X can be any amino acid except proline (Taylor and Drickamer, 2003). The precursor oligosaccharide is assembled on the lipid dolichol inserted into the membrane of the endoplasmic reticulum. The process is initiated in the cytoplasm with the addition of a GlcNAc residue from a UDP-GlcNAc sugar- nucleotide donor to dolichol via a pyrophosphate linkage. This reaction is followed by sequential attachment of another GlcNAc residue followed by five mannose residues to the structure (Hubbard and Ivatt, 1981) (Figure 1.12). At this point the structure flips across the membrane where four mannose residues and three glucose residues are attached to the structure by transfer from mannose and glucose monosaccharides linked to dolichol. The precursor unit is transferred to the protein by the enzyme oliosaccharyltransferase, which acts on asparagine residues that are within the recognition sequences and are exposed at the protein surface in the lumen of the endoplasmic reticulum (Hubbard and Ivatt, 1981; Kornfeld and Kornfeld, 1985).
Once attached to the protein, N-linked glycans are initially processed by removal of the three terminal glucose residues by ER-resident glucosidases. The resulting high-mannose structures are trimmed by mannosidases found in the endoplasmic reticulum and the cis Golgi, which remove some or all of the four mannose residues found in α1-2 linkage. N-linked glycans that do not undergo any further processing than the initial trimming are referred to as high-mannose structures (Kornfeld and Kornfeld, 1985) (Figure 1.12).
The formation of hybrid- and complex-type glycan structures is initiated by the addition of a GlcNAc residue to the α1-3-linked mannose branch. This addition is catalysed by the enzyme GlcNAcTransferase-I (GlcNAcT-I) located in the medial Golgi. Glycans that do not undergo further processing on the α6-mannose arm are referred to as hybrid-type structures (Kornfeld and Kornfeld, 1985) (Figure 1.12). Complex-type N-linked glycans are generated through the action of α-mannosidase II on hybrid-type glycans containing the GlcNAc attached to the α3-mannose branch. This enzyme trims a further two mannose residues from the α6-mannose branch, creating a substrate for GlcNAcT-II to add a GlcNAc to the α6-mannose and resulting in two GlcNAc-terminated branches. Tri- and tetra-antennary glycans are generated through the addition of further GlcNAc residues to one or both of the mannose
Ser/Th -Xxx-A nr s
N
PO4
UDP UMP UDP GDP NH2 S r/ hr-Xxx-Asne T NH2 N N N N N N N N N N N N N N Cytoplasm ER Lumen PO4 Dolichol phosphate UDP Sugar-nucleotide donor mRNA i ii iii iv v vi vii viii ix x N-acetylglucosamine Galactose Glucose Fucose N-acetylneuraminic acid Mannose N N
Figure 1.12 Pathways of N-linked glycosylation. N-linked glycans are assembled as
precursors attached to the lipid dolichol. Synthesis is initiated on the cytoplasmic side of the endoplasmic reticulum (ER) by the sequential addition of two GlcNAc residues followed by five mannose residues (i). Precursors then flip across the membrane to the lumen of the ER (iii), where a further four mannose residues and three glucose residues are added (iv). Completed precursors are attached to asparagine residues of proteins during translocation into the ER lumen (v). Correctly folded proteins are directed for transport out of the ER by the removal of three glucose residues, generating high-mannose type structures (vi). Further trimming of glycans by mannosidases in the Golgi apparatus produce hybrid-type structures (vii) that can either be capped with terminal structures (viii) or can be further processed to generate complex- type structures (ix). Complex glycans can be modified by a family of glycosyltranferases that
branches. GlcNAcT-IV adds a GlcNAc in β1-4 linkage to the α3-mannose branch and GlcNAcT-V adds a GlcNAc in β1-6 linkage to the α6-mannose arm (Varki et al., 1999).
The majority of complex and hybrid-type N-glycans have elongated branches formed by addition of a galactose residue to the GlcNAc in β1-4 linkage by galactosyltransferases resident in the trans Golgi. Sialic acid residues are typically attached to the terminal galactose residue of each branch, which is commonly referred to as ‘capping’ of the branch (Figure 1.12).
Although branches typically consist of only one galactoseβ1-4GlcNAc unit, they can be further lengthened by GlcNActransferases and galactosyltransferases which add repeating GlcNAc and galactose residues referred to as N-acetyllactosamine (LacNAc) sequences. Branches extended with repeating LacNAc sequences are referred to as poly-N-acetyllactosamine chains and consist predominantly of Galβ1- 4GlcNAc repeats, referred to as type II chains. Other less common branch elongations include the addition of galactose in β1-3 linkage, generating a galactoseβ1-3GlcNAc N-acetyllactosamine unit. Chains composed of repeating galactoseβ1-3GlcNAc units are referred to as type I poly-N-acetyllactosamine chains. Typically poly-N-acetyllactosamine extensions occur on only one branch of a glycan, and this is commonly the outer branch of tetra-antennary glycans, where the GlcNAc is attached in β6 linkage to the α6-mannose arm (Varki et al., 1999).
The level of branching, extension of branches and the terminal structures attached to branches all vary depending on the relative expression levels of glycosyltransferases to which the glycan is exposed during its movement through the trans-Golgi. Sialic acid is the most common terminal structure attached to N-linked glycan branches. A family of sialyltransferases catalyse the addition of sialic acid residues to galactose in linkages involving the 3- or 6- hydroxyl group of the galactose, with α2-3 being the most common linkage in humans (Harduin-Lepers et al., 1995).
Another terminal modification involves the attachment of fucose residues to the 3- or 4-hydroxyl groups of GlcNAc and the 2-hydroxyl group of galactose by a family of fucosyltransferases. Glycan structures generated by the attachment of fucose to GlcNAc or galactose are referred to as Lewis structures and are expressed on restricted subsets of cells in the body (Weston et al., 1992).
The enzymatic activities of fucosyl- and sialyltransferases can be affected by previous modifications to a glycan branch. Sialyltransferases have substrate specificity for galactose residues from terminal N-acetyllactosamine branches that have not been fucosylated (Le Pendu et al., 2001; Varki et al., 1999). Many fucosyltransferases can act on internal residues within glycan branches and on terminal sialylated structures. Consequently, glycan branches can contain both terminal sialic acid and fucose (Nishihara et al., 1999).