DISCOVERY AND BACKGROUND

The GlcNAcβ1–Asn linkage was discovered by biochemical analyses of ovalbumin. The minimal amino acid sequence to receive an N-glycan is Asn-X-Ser/Thr in which “X” is any amino acid except Pro. However, not all Asn residues in this sequon are N-glycosylated, as discussed below. Other linkages to Asn include Glc to Asn in laminin of mammals, the S-layer in Archaea and adhesins in some Gram-negative bacteria, GalNAc to Asn in Archaea, and rhamnose or bacillosamine to Asn in bacteria. In a sweet corn glycoprotein, Arg is found in N-linkage to Glc.

N-Glycan synthesis begins on a lipid-like polyisoprenoid molecule termed dolichol-phosphate (Dol-P) in eukaryotes. Following synthesis of an oligosaccharide that contains as many as 14 sugars, the N-glycan is transferred “en bloc” to protein. This synthetic pathway is conserved in all metazoa, plants, and yeast. Bacteria use related mechanisms to synthesize cell wall (Chapter 21). N-Glycans affect many properties of glycoproteins including their conformation, solubility, antigenicity, activity, and recognition by glycan-binding proteins (GBPs). Introduction of an N-glycan site (Asn-X-Ser/Thr) is used as a method to localize or orient a glycoprotein in the secretory pathway, or to follow its trafficking through the cell. Defects in N-glycan synthesis lead to a variety of human diseases (Chapter 45).

MAJOR CLASSES AND NOMENCLATURE IN EUKARYOTES

All eukaryotic N-glycans share a common core sequence, Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1–4GlcNAcβ1–Asn-X-Ser/Thr, and are classified into three types: (1) oligomannose, in which only Man residues extend the core; (2) complex, in which “antennae” initiated by GlcNAc extend the core; and (3) hybrid, in which Man extends the Manα1-6 arm of the core and one or two GlcNAc-initiated antennae extend the Manα1-3 arm (Figure 9.1).

FIGURE 9.1.

Types of N-glycans. N-Glycans at Asn-X-Ser/Thr sequons in eukaryote glycoproteins are of three general types: oligomannose, complex, and hybrid. Each N-glycan contains the common core Man₃GlcNAc₂Asn. Complex N-glycans can have up to five branches initiated (more...)

PREDICTING SITES OF N-GLYCOSYLATION IN EUKARYOTES

N-Glycans are added to secreted and membrane-bound glycoproteins at Asn-X-Ser/Thr sequons. About 70% of proteins contain this sequon, and ∼70% of sequons carry an N-glycan. Mapping of the murine N-glycoproteome revealed more than 10,000 different N-glycosylation sites. Occasionally, N-glycans occur at Asn-X-Cys or, rarely, with a different amino acid in the third position. The transfer of an N-glycan to Asn-X-Ser/Thr occurs on the lumenal side of the endoplasmic reticulum (ER) membrane, during or after the translocation of the protein substrate. There is no definitive evidence that N-glycans occur on cytoplasmic or nuclear proteins nor on the cytoplasmic portions of membrane proteins. However, N-glycans covalently attached to RNA and expressed at the cell surface have recently been discovered. In proteins, only Asn-X-Ser/Thr sequons accessible to the ER lumen are known to receive an N-glycan. “X” may not be a Pro or may reduce the efficiency of glycosylation, such as when “X” is acidic (Asp or Glu), or enhance the efficiency, such as when Phe is in an adjacent reverse turn. However, although the presence of Asn-X-Ser/Thr is necessary for the receipt of an N-glycan, transfer does not always occur, because of conformational or other constraints during glycoprotein folding. Thus, Asn-X-Ser/Thr sequons encoded by an mRNA are referred to as potential N-glycan sites. Proof that an N-glycan is actually present requires experimental evidence, as described later in this chapter.

ISOLATION, PURIFICATION, AND ANALYSIS

N-Glycans of eukaryotes may be released from Asn using the bacterial enzyme peptide-N-glycosidase F (PNGase F). This enzyme will remove oligomannose, hybrid, and complex N-glycans attached to Asn unless the N-glycan core has certain modifications found in slime molds, plants, insects, and parasites. Another enzyme termed PNGase A (from almonds) will remove all N-glycans. Both enzymes are amidases that release N-glycans attached to the nitrogen of Asn, thereby converting Asn to Asp. Therefore, sites of glycosylation can be deduced by amino acid sequence analysis performed before and after PNGase F or A treatment. Other bacterial enzymes cleave between the two GlcNAc residues of the N-glycan core, leaving one GlcNAc attached to Asn. Endoglycosidase H releases oligomannose and hybrid N-glycans, but not complex N-glycans. Endoglycosidase F1 is similar to endoglycosidase H, whereas endoglycosidase F2 releases primarily biantennary N-glycans, and endoglycosidase F3 releases bi- and triantennary N-glycans, with a preference for those with a Fuc residue in the core. N-Glycans may also be released by hydrazinolysis or by exhaustive digestion with a protease that removes all amino acids except for the Asn. Released N-glycans may be purified by conventional ion-exchange and size-exclusion chromatography, high-performance liquid chromatography (HPLC) methods, and affinity chromatography on GBPs such as lectins. Lectins for glycan analysis are usually obtained from plants (Chapter 48). Release of N-glycans using chemical and enzymic methods, purification, and analysis are described in Chapters 50 and 51.

SYNTHESIS OF N-GLYCANS IN EUKARYOTES

N-Glycan biosynthesis occurs in two phases and in two compartments of eukaryotic cells, the ER and the Golgi (Chapter 4). The first phase is a highly conserved pathway that proceeds at the ER membrane on the lipid carrier dolichol-phosphate (Dol-P). An oligosaccharide assembled on Dol-P is transferred to Asn in select Asn-X-Ser/Thr sequons of secretory and membrane proteins during their translocation into the ER. The second phase begins with processing of N-glycans by glycosidases and glycosyltransferases in the lumen of the ER and continues in the Golgi in a species-, cell type–, protein-, and even site-specific manner. Many of the glycosidases and glycosyltransferases are differentially expressed and their activity is exquisitely sensitive to the physiological state of the cell. All glycosyltransferases use activated sugars (nucleotide sugars, dolichol sugars) as substrates (Chapter 5). Thus, a mature glycoprotein carries N-glycans that depend on the complement of expressed glycosylation genes in the cell type in which the glycoprotein is made and on the physiological state of that cell that may affect the localization and activity of glycosylation enzymes and nucleotide sugar transporters.

Synthesis of the Dolichol-Linked Precursor

Dolichol is a polyisoprenoid comprised of five-carbon isoprene units (Figure 9.2). The most common yeast dolichol has 14 isoprene units, whereas dolichols from other eukaryotes, including mammals, may have up to 19 isoprene units. The structure of the mature N-glycan precursor synthesized on Dol-P is shown in Figure 9.3. Genetic studies in Saccharomyces cerevisiae have identified conserved ALG (Asn-linked glycosylation) loci that encode the biosynthetic machinery for the assembly of the lipid-linked oligosaccharide in eukaryotes (Figure 9.3).

FIGURE 9.2.

Dolichol phosphate (Dol-P). N-Glycan synthesis begins with the transfer of GlcNAc-1-P from UDP-GlcNAc to Dol-P to generate dolichol pyrophosphate N-acetylglucosamine (Dol-P-P-GlcNAc). This reaction is inhibited by tunicamycin, which blocks N-glycan synthesis. (more...)

FIGURE 9.3.

Synthesis of Dolichol-P-P-GlcNAc₂Man₉Glc₃. Dolichol (red squiggle) phosphate (Dol-P) located on the cytoplasmic face of the endoplasmic reticulum (ER) membrane receives GlcNAc-1-P from UDP-GlcNAc in the cytoplasm to generate Dol-P-P-GlcNAc. Dol-P-P-GlcNAc (more...)

The first step is catalyzed by ALG7 (DPAGT1 in mammals), a GlcNAc-1-phosphotransferase that transfers GlcNAc-1-P from UDP-GlcNAc to form Dol-P-P-GlcNAc. Tunicamycin, an inhibitor of this enzyme, is used to inhibit N-glycosylation in cells. A second GlcNAc and five Man residues are subsequently transferred from UDP-GlcNAc and GDP-Man, respectively, to generate Man₅GlcNAc₂-P-P-Dol on the cytoplasmic side of the ER membrane (Figure 9.3). All of these enzymes transfer only the sugar portion of the nucleotide sugar. The Man₅GlcNAc₂-P-P-Dol precursor translocates across the ER membrane bilayer via a “flippase” encoded by the RFT1 locus in yeast. Man₅GlcNAc₂-P-P-Dol is extended by the addition of four Man and three Glc residues transferred from Dol-P-Man and Dol-P-Glc, respectively. Dol-P-Man and Dol-P-Glc donors are formed on the cytoplasmic side of the ER membrane from GDP-Man and UDP-Glc. Dol-P-Man and Dol-P-Glc must also be flipped across the ER membrane. Mammalian MPDU1 is an ER membrane protein necessary for the utilization of Dol-P-Man and Dol-P-Glc in the ER lumen in the synthesis of the mature N-glycan precursor Glc₃Man₉GlcNAc₂-P-P-Dol (Figure 9.3). This 14-sugar glycan is transferred by oligosaccharyltransferase (OST) to Asn in receptive Asn-X-Ser/Thr sequons in protein regions that have translocated across the ER membrane.

Early Processing Steps: Glc₃Man₉GlcNAc₂Asn to Man₅GlcNAc₂Asn

Following the covalent attachment of the 14-sugar glycan to Asn-X-Ser/Thr in a protein, processing reactions trim the N-glycan in the ER. The initial steps have key roles in regulating glycoprotein folding via interactions with ER chaperones that recognize specific features of the trimmed N-glycan (Chapter 39). Processing of Glc₃Man₉GlcNAc₂Asn begins with the sequential removal of Glc residues by α-glucosidases I (MOGS) and II (GANAB) (Figure 9.4). Both glucosidases function in the lumen of the ER, with α-glucosidase I acting specifically on the terminal α1–2Glc and α-glucosidase II sequentially removing the two inner α1–3Glc residues. Removal of Glc residues, and the transient re-addition of the innermost α1–3Glc during protein folding, contribute to ER retention time. The removal of Glc may be prevented experimentally by the use of glucosidase I inhibitors such as castanospermine or deoxynojirimycin (Chapter 55). Following inhibition, N-glycans retain the three Glc residues and usually lose one or two Man residues as they pass through the ER and Golgi, resulting in Glc₃Man_7–9 GlcNAc₂ N-glycans on mature glycoproteins. Before exiting the ER, ER α-mannosidase I (MAN1B1), removes the terminal α1-2Man from the central arm of Man₉GlcNAc₂ to yield a Man₈GlcNAc₂ isomer (Figure 9.4). Slow cleavage of terminal α1-2Man residues on misfolded glycoproteins by EDEM mannosidases leads to recognition of the trimmed glycans by OS9 and targets them for ER degradation (Chapter 39). The majority of glycoproteins exiting the ER to the Golgi carry N-glycans with either eight or nine Man residues.

FIGURE 9.4.

Processing and maturation of an N-glycan. The mature glycan attached to Dol-P-P (Figure 9.3) is usually transferred to Asn-X-Ser/Thr sequons during protein synthesis as proteins are being translocated into the endoplasmic reticulum (ER). Some transfer (more...)

Some N-glycans in the cis-Golgi retain a Glc residue because of incomplete processing in the ER. In this case, Golgi endo-α-mannosidase (MANEA) cleaves internally between the two Man residues of the Glcα1-3Manα1-2Manα1-2 moiety, thereby generating a Man₈GlcNAc₂ isomer different from that produced by ER α-mannosidase I (MAN1B1). Trimming of α1-2Man residues continues with the action of α1-2 mannosidases IA, IB, and IC (MAN1A1, MAN1A2, MAN1C1) in the cis-Golgi to give Man₅GlcNAc₂ (Figure 9.4) to yield Man₅GlcNAc₂, a key intermediate in the pathway to hybrid and complex N-glycans (Figure 9.1). Some Man_5-9GlcNAc₂ glycans may also escape further modification. In these cases, a mature membrane or secreted glycoprotein will carry Man_5–9GlcNAc₂ N-glycans. In addition, the action of MAN1B1, MAN1A1, MAN1A2, and MAN1C1 can all be blocked experimentally by the inhibitors, deoxymannojirimycin or kifunensine, resulting in Man_8-9GlcNAc₂ on mature glycoproteins. Most mature glycoproteins have some oligomannose N-glycans that are not processed in the cis-Golgi.

Late Processing Steps: Man₅GlcNAc₂Asn to Hybrid and Complex N-Glycans

Biosynthesis of hybrid and complex N-glycans (Figure 9.1) is initiated in the medial-Golgi by the action of an N-acetylglucosaminyltransferase called GlcNAc-TI (MGAT1), which adds a GlcNAc residue to the C-2 of the α1-3Man in the core of Man₅GlcNAc₂ (Figure 9.4). Subsequently, the majority of N-glycans are trimmed by α-mannosidase II enzymes (MAN2A1 or MAN2A2) in the medial-Golgi, which remove the terminal α1-3Man and α1-6Man residues from GlcNAcMan₅GlcNAc₂ to form GlcNAcMan₃GlcNAc₂. It is important to note that α-mannosidase II cannot trim Man₅GlcNAc₂ unless it has been acted on by MGAT1. Once both Man residues are removed, a second GlcNAc is added to the C-2 of the α1-6Man in the N-glycan core by the action of GlcNAc-TII (MGAT2) to yield the precursor for all biantennary, complex N-glycans. Hybrid N-glycans are formed if the GlcNAcMan₅GlcNAc₂ glycan produced by MGAT1 is not acted on by α-mannosidase II. Incomplete action of α-mannosidase II can result in GlcNAcMan₄GlcNAc₂ hybrids. Small oligomannose N-glycans have been found in relatively large amounts in invertebrates and plants. These Man_3–4GlcNAc₂ N-glycans (paucimannose N-glycans) are formed from GlcNAcMan_3–4GlcNAc₂ following removal of the peripheral GlcNAc by a Golgi hexosaminidase that acts after α-mannosidase II (Chapters 24 and 26). Paucimannose glycans are also found in mammals, and can be elevated in cancer, inflammation, and stem cell development.

The complex N-glycan shown in the medial-Golgi of Figure 9.4 has two antennae or branches initiated by the addition of two GlcNAc residues. Additional branches can be initiated at C-4 of the core α1-3Man by GlcNAc-TIV (MGAT4A, MGAT4B, MGAT4C) and at C-6 of the core α1-6Man by GlcNAc-TV (MGAT5) to yield tri- and tetra-antennary N-glycans (Figure 9.5). MGAT5B or GlcNAc-TIX catalyzes the same reaction but preferentially on O-mannose glycans in brain. Another branch, found in birds and fish, can be initiated at C-4 of the core α1-6Man by GlcNAc-TVI (MGAT6; Figure 9.5). Genes related to GlcNAc-TVI exist in mammalian genomes (MGAT4C). Complex and hybrid N-glycans may also carry a “bisecting” GlcNAc residue that is attached to the β-Man of the core by GlcNAc-TIII (MGAT3) (Figure 9.5). A bisecting GlcNAc on a biantennary N-glycan is shown in Figure 9.5, and it may be present in all of the more highly branched N-glycans. However, it is not usually extended.

FIGURE 9.5.

Branching and core modification of complex N-glycans. The hybrid and mature, biantennary, complex N-glycans shown in Figure 9.4 may contain more branches because of GlcNAc-transferases in the Golgi that act only after MGAT1 has acted. If the α-mannosidase (more...)

Maturation of N-Glycans

Further sugar additions convert the limited repertoire of hybrid and branched N-glycans into an extensive array of mature, complex N-glycans comprising (1) sugar additions to the N-glycan core, (2) elongation of branching GlcNAc residues by sugar additions, and (3) “capping” or “decoration” of elongated branches.

The major core modification in vertebrate N-glycans is the addition of α1-6Fuc to the Asn-linked GlcNAc in the N-glycan core (Figure 9.5). The α1-6 fucosyltransferase (FUT8) usually requires the prior action of MGAT1 (Figure 9.4). In invertebrate glycoproteins, both core GlcNAc residues may receive a Fuc in an α1–3 and/or α1-6 linkage (Chapters 25 and 26). In plants, Fuc is transferred to the Asn-linked GlcNAc only in α1-3 linkage (Chapter 24). Also, in plant and helminth glycoproteins, the addition of β1-2Xyl to the β-Man of the core is common. This xylosyltransferase also requires the prior action of MGAT1. Xylose has not been detected in vertebrate N-glycans.

The majority of complex and hybrid N-glycans have extended branches that are made by the addition of Gal to the initiating GlcNAc to produce the ubiquitous building block Galβ1-4GlcNAc, referred to as a type-2 N-acetyllactosamine or “LacNAc” sequence (Figure 9.6). The sequential addition of LacNAc disaccharides gives tandem repeats termed poly-LacNAc. In some glycoproteins, β-linked GalNAc is added to GlcNAc instead of Gal, yielding antennae with a GalNAcβ1–4GlcNAc (LacdiNAc) extension (Chapter 14).

FIGURE 9.6.

Typical complex N-glycans found on mature glycoproteins. A LacNAc unit (bracketed) on any branch may be repeated many times.

The most important “capping” reactions involve the addition of Sia, Fuc, Gal, GlcNAc, and sulfate to complex N-glycan branches. Capping sugars are most commonly α-linked and therefore protrude away from the β-linked poly-LacNAc branches, thus facilitating the presentation of terminal sugars to lectins and antibodies. Many of these structures are shared by N- and O-glycans and by glycolipids (Chapter 14). Terminal sialic acids can have further chemical modifications (e.g., O-acetylation) (Chapter 15).

The various reactions described above potentially yield a myriad of complex N-glycans that differ in branch number, composition, length, capping arrangements, and core modifications. Some examples to illustrate this diversity are shown in Figure 9.6.

THE PHOSPHORYLATED N-GLYCANS ON LYSOSOMAL HYDROLASES

Lysosomal hydrolases degrade proteins, lipids, and glycans in the lysosome. Many of these enzymes are targeted to lysosomes by a specialized trafficking pathway that requires phosphorylated oligomannose N-glycans. The phosphorylation step involves the transfer of GlcNAc-1-P to C-6 of Man residues of oligomannose N-glycans on lysosomal hydrolases in the cis-Golgi by N-acetylglucosamine-1-phosphotransferase (heterodimer of GNPTAB and GNPTG) (Figure 9.4). A phosphodiester N-acetylglucosamindase (NAGPA) in the trans-Golgi removes the GlcNAc to generate Man-6-P recognized by a Man-6-P receptor (M6PR). M6PRs transport lysosomal hydrolases to an acidified compartment which ultimately fuses with the lysosome. The details of this trafficking pathway are presented in Chapter 33.

TRANSFERASES AND TRANSPORTERS IN N-GLYCAN SYNTHESIS

The glycosyltransferases in the ER are mainly multitransmembrane proteins woven into the ER membrane. In contrast, the glycosyltransferases in Golgi compartments are generally type II membrane proteins with a small cytoplasmic amino-terminal domain, a single transmembrane domain, and a large lumenal domain that includes an elongated stem region extending from the membrane, and a globular catalytic domain (Chapter 6). The stem region is often cleaved by signal peptide peptidase-like proteases, particularly SPPL-3, releasing the catalytic domain into the lumen of the Golgi and allowing its secretion. Thus, extracellular soluble forms of many glycosyltransferases exist in tissues and sera and may function as transferases if nucleotide sugars are present extracellularly. Nucleotide sugars are synthesized in the cytoplasm, except for CMP-sialic acids, which are synthesized in the nucleus (Chapter 5). They are subsequently concentrated in the appropriate compartment following transport across the membrane by specialized nucleotide sugar transporters that translocate CMP-Sia, UDP-Gal, UDP-Glc, UDP-GlcNAc, GDP-Fuc, and other nucleotide sugars. A few of these transporters can transport more than one nucleotide sugar. Each transporter is a multitransmembrane protein that usually contains 10 membrane-spanning domains. Some CMP-sialic acids can be further modified within the lumen of the Golgi by O-acetyl groups before transfer to a glycan acceptor (Chapter 15).

GLYCOPROTEINS COMPRISE MANY GLYCOFORMS

Glycoproteins usually have a range of different N-glycans on a particular Asn-X-Ser/Thr N-glycosylation sequon, leading to glycan heterogeneity at each site. Furthermore, when there is more than one Asn-X-Ser/Thr sequon per molecule, different molecules in a population may have different subsets of N-glycans on different sequons, leading to glycoprotein microheterogeneity. Glycoproteins that differ only in their N-glycan complement are termed glycoforms. The variation in N-glycans of a glycoprotein may be due to protein conformation affecting substrate availability for Golgi glycosidases or glycosyltransferases, nucleotide sugar metabolism, transport rate of the glycoprotein through the lumen of the ER and Golgi, and the proximity of an Asn-X-Ser/Thr sequon to a transmembrane domain. Also, localization of glycosyltransferases within subcompartments of the Golgi can determine which enzymes encounter N-glycan acceptors. It is important to note that glycosylation enzymes often compete for the same acceptor and that most glycosyltransferases and glycosidases require the prior actions of other glycosyltransferases and glycosidases before they can act (Figure 9.4).

FUNCTIONS OF N-GLYCANS

Determining the functions of N-glycans may be accomplished using inhibitors including tunicamycin, which blocks the first step of N-glycosylation, or castanospermine, deoxynojirimycin, deoxymannojirimycin, kifunensine, and swainsonine, which block N-glycan processing; or glycosylation mutants of model organisms such as yeast, cultured mammalian cells, Drosophila melanogaster, Caenorhabditis elegans, zebrafish, and mouse. The various chemical inhibitors of N-glycan synthesis are discussed in Chapter 55. Many yeast mutants in the synthesis and initial processing of N-glycans are identified in Figure 9.3, and three mutants of cultured cells with altered glycosylation are identified in Figure 9.4 and described in detail in Chapter 49. Mutant cells or organisms with an altered N-glycosylation ability provide enormous insights into the biological functions of N-glycans, and their contributions to the biochemical properties of a glycoprotein in terms of structure, activity, susceptibility to proteases, and antigenicity. In addition, mutant cells and organisms allow glycosylation pathways that operate in vivo to be defined. A cell or organism with a loss-of-function mutation usually accumulates the biosynthetic intermediate that is the substrate of the activity lost in the mutant. Gain-of-function mutations reveal alternative pathways or glycosylation reactions that may occur depending on cell type. N-glycan functions have also been determined from the features of human congenital disorders of glycosylation (CDG) (Chapter 45).

Mouse mutants, in particular, have provided enormous insights into the functions of individual sugars present in N-glycans, as well as the functions of whole classes of N-glycans. Thus, deletion of the Mgat1 gene that encodes MGAT1 prevents the synthesis of complex and hybrid N-glycans so that Man₅GlcNAc₂ is found at all complex and hybrid N-glycan sites (Figure 9.4). Whereas the absence of MGAT1 does not affect the viability or growth of Lec1 cultured cells under normal conditions, elimination of MGAT1 in the mouse results in death during embryonic development (Chapter 41). The complex N-glycans are important in retaining growth factor and cytokine receptors at the cell surface, probably through interactions with GBPs such as galectins, or cytokines such as transforming growth factor-β. Deletion of genes encoding sialyltransferases, fucosyltransferases, or branching N-acetylglucosaminyltranferases other than MGAT1 has generally produced viable mice with defects in immunity or neuronal cell migration, emphysema, or inflammation. N-Glycans may carry the sugar determinants recognized by selectins that mediate cell–cell interactions important for leukocyte extravasation from the bloodstream and regulation of lymphocyte homing to lymph nodes (Chapter 34). N-Glycans are known to become more branched when cells become cancerous, and this change facilitates cancer progression (Chapter 47). Tumors formed in mice lacking MGAT5 are retarded in their progression. Thus, certain glycosyltransferases may be appropriate targets for the design of cancer therapeutics.

ACKNOWLEDGMENTS

The authors acknowledge the contributions of Harry Schachter to previous versions of this chapter and appreciate helpful comments and suggestions from Thierry Hennet to this version.

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