Chapter  5:  Glycosyltransferases and Glycan-processing Enzymes

GENERAL PROPERTIES

FIGURE 5.1

Glycosylation reactions. A glycosyltransferase uses (more...) Download Teaching Slide

FIGURE 5.1

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Glycosylation reactions. A glycosyltransferase uses a glycosyl donor and an acceptor substrate. In animals, glycosyl donors include nucleotide sugars and dolichol-phosphate-linked monosaccharides and oligosaccharides. Bacteria also use undecaprenyl-pyrophosphate (PP)-linked donors. Acceptors are most commonly oligosaccharides, but (in rare cases) they can be monosaccharides. Proteins and ceramides are also acceptors for the glycosyltransferases that initiate glycoprotein, proteoglycan, and glycolipid synthesis. Many other targets, such as drugs and other small molecules, can be glycosylated, but these are not discussed in this chapter. Even DNA can be glycosylated.

Figure 5.1

The glycosyltransferases that initiate the synthesis of glycoconjugates use acceptor substrates that include oligosaccharides, monosaccharides, polypeptides, lipids, small organic molecules, and even DNA. Only enzymes involved in the biosynthesis of glycoproteins, proteoglycans, and glycolipids are discussed in this chapter. The vast majority of these glycosyltransferases are responsible for elongating glycan chains. Generally speaking, these enzymes act sequentially, so that the product of one enzyme yields a preferred acceptor substrate for the subsequent action of another. The end result is a linear and/or branched polymer composed of monosaccharides linked to one another. In most cases, acceptor recognition does not involve the underlying polypeptide or glycolipid substrate, but several notable exceptions exist that are described below. Despite this finding, specific glycosylation sites of many glycoproteins carry predictable glycan structures, suggesting a complex mechanism for controlling composition.

FIGURE 5.2

Glycan-modifying enzymes. A variety of donors are (more...) Download Teaching Slide

FIGURE 5.2

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Glycan-modifying enzymes. A variety of donors are used to modify glycans.

Figure 5.2

GLYCOSYLTRANSFERASE SPECIFICITY

FIGURE 5.3

Strict acceptor substrate specificity of glycosyltransferases (more...) Download Teaching Slide

FIGURE 5.3

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Strict acceptor substrate specificity of glycosyltransferases as exemplified by the human B blood group α1–3 galactosyltransferase. The B transferase adds galactose in α1–3 linkage to the H antigen (top). This enzyme requires the α1–2-linked fucose modification of the H antigen for activity because the B transferase does not add to an unmodified type-2 precursor (middle), or precursors modified by sialyl residues (bottom) or other monosaccharides (not shown). (For the monosaccharide symbol code, see Figure 1.5, which is reproduced on the inside front cover.)

Symbol Key:

Figure 5.3

Figure 5.3

We now know that there are also instances in which more than one enzyme can use the same acceptor to make the same linkage. The human fucosyltransferases III–VII, for example, all attach fucose in α1–3 linkage to N-acetyllactosamine moieties on glycans (see Chapter 13). Other examples include α2–3 sialyltransferases that act on galactose, β1–4 galactosyltransferases that act on N-acetylglucosamine, and the family of GlcNAc 3-O-sulfotransferases that participate in heparin/heparan sulfate synthesis. In some rare cases, a single enzyme can catalyze more than one reaction. Human fucosyltransferase III can attach fucose in either α1–3 or α1–4 linkage, and an enzyme called EXTL2 can attach either N-acetylgalactosamine or N-acetylglucosamine in α linkage to glucuronic acid. The β1–4 galactosyltransferase involved in N-acetyllactosamine formation exhibits an unusual flexibility in specificity. When β1-4 galactosyltransferase binds α-lactalbumin (the complex is called lactose synthase), it switches its acceptor specificity from N-acetylglucosamine to glucose, which enables lactose synthesis during milk formation.

Finally, some glycosyltransferases (such as those that synthesize the backbones of glycosaminoglycans) have two separate active sites, for example, one that catalyzes the attachment of N-acetylglucosamine to glucuronic acid and another that attaches glucuronic acid to N-acetylglucosamine (see Chapters 15 and 16). However, the examples described above are all exceptions to the generally strict donor, acceptor, and linkage specificity exhibited by most glycosyltransferases, a property that serves to define and limit the number and type of glycan structures observed in a given organism.

TABLE 5.1

Amino acid–consensus sequences or glycosylation (more...)

TABLE 5.1

Amino acid–consensus sequences or glycosylation motifs for the formation of glycopeptide bonds

Glycopeptide bond	Consensus sequence or peptide motif
GlcNAc-β-Asn	Asn-X-Ser/Thr (X = any amino acid except Pro)
Glc-β-Asn	Asn-X-Ser/Thr
GalNAc-α-Ser/Thr	repeat domains rich in Ser, Thr, Pro, Gly, Ala in no special sequence
GlcNAc-α-Thr	Thr-rich domain near Pro residues
GlcNAc-β-Ser/Thr	Ser/Thr-rich domains near Pro, Val, Ala, Gly
Man-α-Ser/Thr α	Ser/Thr-rich domains
Fuc-α-Ser/Thr	EGF modules (Cys-X-X-Gly-Gly-Thr/Ser-Cys) TSR modules (TrpX5CysX2-3Ser/ThrCysX2Gly)
Glc-β-Ser	EGF modules (Cys-X-Ser-X-Pro-Cys)
Xyl-β-Ser	Ser-Gly (in the vicinity of one or more acidic residues)
Glc/GlcNAc-Thr	Rho: Thr-37; Ras, Rac; Cdc42: Thr-35
Gal-Thr	Gly-X-Thr (X = Ala, Arg, Pro, Hyp, Ser) (vent worm)
Gal-β-Hyl	collagen repeats (X-Hyl-Gly)
Ara-α-Hyp	repetitive Hyp-rich domains (e.g., Lys-Pro-Hyp-Hyp-Val)
GlcNAc-Hyp	Skp1: Hyp-143
Glc-α-Tyr	glycogenin: Tyr-194
GlcNAc-α-1-P-Ser	Ser-rich domains (e.g., Ala-Ser-Ser-Ala)
Man-α-1-P-Ser	Ser-rich repeat domains
Man-α-C-Trp	Trp-X-X-Trp
Man-6-P-ethanolamine-protein	GPI attached after cleavage of carboxy-terminal peptide

Based on Spiro R.G. 2002. Glycobiology 12: 43R–56R; see also Table 1.7.

(EGF) Epidermal growth factor; (TSR) thrombospondin repeats; (Hyp) hydroxyproline; (Hyl) hydroxylysine; (GPI) glycosylphosphatidylinositol; (Ara) arabinose.

PROTEIN/GLYCOPROTEIN ACCEPTORS AND GLYCOSYLTRANSFERASE ACTION

Among the glycosyltransferases that use proteins/glycoproteins as acceptor substrates, the underlying polypeptide chain of the acceptor is used in different ways to confer specificity on the glycosylation reaction. As mentioned above, all N-glycans are initiated by oligosaccharyltransferase, an enzyme that cotranslationally transfers the N-glycan precursor to asparagine residues in the sequence motif Asn-X-Ser/Thr. In contrast, many enzymes can glycosylate serine and threonine residues leading to O-glycans possessing O-GlcNAc, O-GalNAc, O-fucose, O-mannose, and O-xylose linkages. Among the enzymes responsible for these linkages, specificity for a particular serine or threonine residue is achieved in different ways. For example, O-xylosylation has an absolute requirement for a glycine residue located amino terminally to the serine with nearby acidic residues on one or both sides of the glycosylation site. Some polypeptide O-GalNAc transferases even possess a lectin domain that serves to direct the glycosyltransferase to regions of polypeptide already possessing glycan chains. In this way, regions of polypeptide that have a high degree of carbohydrate substitution, typical of mucin structures, can be synthesized.

FIGURE 5.4

Recognition site for glycoprotein hormone N-acetylgalactosaminyltransferase (more...) Download Teaching Slide

FIGURE 5.4

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Recognition site for glycoprotein hormone N-acetylgalactosaminyltransferase on human chorionic gonadotropin. Ribbon diagram of a fragment (residues 34–58) of human chorionic gonadotropin (PDB [protein data bank] ID 1hrp). The Pro40-Leu41-Arg42 tripeptide and residues Lys44 and Lys45 correspond to residues essential for recognition by glycoprotein hormone N-acetylgalactosaminyltransferase. Residues Asn52-Val53-Thr54 correspond to the N-glycosylation sequence motif of the N-glycan (at Asn52) modified by the glycoprotein hormone N-acetylgalactosaminyltransferase. Only the chitobiose core (GlcNAcβ1–4GlcNAc) of the acceptor N-glycan is shown.

Figure 5.4

In a variation on this theme, GlcNAc-1-phosphotransferase is able to modify a family of enzymes that are specifically destined for lysosomes (see Chapter 30). In this case, lysine residues appropriately spaced and positioned relative to the N-glycan being modified have been shown to be important determinants of acceptor specificity.

The ER resident glucosyltransferase (GGT) has an acceptor substrate specificity unique among glycosyltransferases. It specifically reglucosylates N-glycans on misfolded glycoproteins, rendering them substrates for the chaperones calnexin and calreticulin (see Chapter 36). Its ability to differentiate between misfolded and properly folded glycoproteins is a critical component of the ER quality control machinery.

GLYCOSYLTRANSFERASE SEQUENCE FAMILIES AND FOLD TYPES

FIGURE 5.5

Sialyl motifs. Domain structure of a typical sialyltransferase, (more...) Download Teaching Slide

FIGURE 5.5

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Sialyl motifs. Domain structure of a typical sialyltransferase, showing the sialyl motifs shared by this family of enzymes. The sialyl L motif of 48–49 amino acids shares significant similarity among members and may be up to 65% identical in amino acid sequence. The sialyl S motif is smaller (~23 amino acids) and diverges more among members of the family, with only two absolutely conserved residues. In both cases, identical residues are indicated and residues showing similarity are denoted by parentheses. (Asterisk) Position of a highly conserved sequence H-X₄-E. Additional conserved motifs may exist as well.

Symbol Key:

Figure 5.5

FIGURE 5.6

Ribbon diagrams of representative GT-A and GT-B folds. (more...) Download Teaching Slide

FIGURE 5.6

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Ribbon diagrams of representative GT-A and GT-B folds. The GT-A and -B structures correspond to those of rabbit β1-2 N-acetylglucosaminyltransferase I (PDB ID 1foa) and T4 phage β-glucosyltransferase (PDB ID 1j39), respectively. In both cases, bound nucleotide sugar donor substrate is shown in stick representation.

Figure 5.6

CATALYTIC MECHANISMS

FIGURE 5.7

Schematic representation of (a) inverting and (more...) Download Teaching Slide

FIGURE 5.7

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Schematic representation of (a) inverting and (b) retaining catalytic mechanisms. (a) S_N2-like attack of the acceptor leads to inversion of stereochemistry at C1. For a glycosidase reaction, R2 would correspond to a proton and R1 would be the remainder of the glycan. A and B label general acid and base groups in the catalytic site of the enzyme. For a glycosyltransferase reaction, R2 would correspond to the remainder of the acceptor substrate and R1 would typically be the nucleoside monophosphate or diphosphate moiety of the donor substrate. (b) This mechanism has only been established for glycosidases. Two successive S_N2-like reactions separated by a glycosyl-enzyme intermediate lead to retention of the configuration at C1. R1 corresponds to the remainder of the glycan and R2 to a proton.

FIGURE 5.8

Catalytic site of bovine β1–4 galactosyltransferase. (more...) Download Teaching Slide

FIGURE 5.8

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Catalytic site of bovine β1–4 galactosyltransferase. Composite figure shows selected residues/atoms of the superimposition of the donor complex (PDB ID 1tw1) on the acceptor complex (PDB ID 1tw5). O4 designates the C4 hydroxyl group of the GlcNAcβ1–4GlcNAc acceptor substrate positioned for in-line S_N2 attack (arrow) on C1 of the UDP-Gal donor substrate. The base form of D318 serves to partially deprotonate the C4 hydroxyl group rendering it a better nucleophile. The positively charged Mg⁺⁺ ion coordinates the two phosphates of the UDP leaving group, promoting cleavage of the C1-P_β bond by stabilizing the additional negative charge that develops on P_β of the leaving group. D252-V253-D254 corresponds to the DXD motif in bovine β1–4 galactosyltransferase.

Figure 5.7

Figure 5.7a

Figure 5.8

Figure 5.8

Figure 5.7a

Figure 5.7b

Despite similarities in the mechanisms and transition states used by glycosidases and glycosyltransferases, small-molecule inhibitors of many glycosidases exist (see Chapter 50), whereas there are relatively few glycosyltransferase inhibitors. This probably reflects the fact that many good enzyme inhibitors mimic the transition state, a relatively simple task in the case of the glycosidase reaction where the acceptor substrate is a simple water molecule. Simple high-affinity competitive inhibitors of substrate binding provide another means of inhibiting an enzyme reaction (see Chapter 50).

KINETIC MECHANISMS

In addition to understanding the structure and catalytic mechanisms of glycosyltransferases, there is much interest in understanding the kinetic =mechanism. Many glycosyltransferases have been shown to possess a Bi Bi sequential kinetic mechanism in which the donor substrate binds before the acceptor substrate, and the glycosylated acceptor is released before the nucleoside monosphosphate or disphosphate, depending on the reaction. Such kinetics are easily explained by a structural model in which the active site represents a deep pocket, with the nucleotide sugar substrate at the bottom and the acceptor substrate stacked on top. If the acceptor substrate were to bind first, it would sterically preclude donor substrate binding. Necessarily, release of the glycosylated product must precede release of the nucleoside phosphate. Although largely consistent with such a model, the X-ray crystal structures of glycosyltransferase-substrate complexes also shows that substrate-dependent ordering of flexible loops is a feature common to glycosyltransferases. Typically, donor substrate binding orders a loop(s) that in turn facilitates acceptor substrate binding.

SULFATION AND OTHER MODIFICATIONS

Glycans can undergo a number of modifications, depending on the major subclass and species of origin. The sulfotransferases are a large family of enzymes found in both the cytoplasm and the Golgi. Enzymes from the cytoplasm sulfate low-molecular-weight substrates such as steroids and exogenous compounds, whereas those from the Golgi sulfate a large number of cell-surface and secreted glycans. Sulfotransferases have a particularly important role in the production of glycosaminylglycans, molecules involved in embryological development and physiology (see Chapters 16 and 24). They are also involved in the formation of L-selectin ligands, glycans required for the trafficking of lymphocytes across high endothelial venules in lymph nodes (see Chapter 31). All sulfotransferases use 3′-phospho-adenosine-5′-phosphosulfate (PAPS) as the sulfate donor (see Chapter 4). Although the sequence similarity among sulfotransferases can be very low, they all possess conserved sequence motifs responsible for binding the 5′ and 3′ phosphate groups of PAPS. Moreover, structural analysis has shown that all of the sulfotransferases solved to date possess the same basic structure. Mechanistically, the enzyme proceeds via an S_N2-like reaction, with the hydroxyl group of the acceptor making an in-line attack on the sulfate group. In conjunction with structural analysis, mutagenesis has provided insight into the catalytic role played by a number of residues highly conserved among the sulfotransferases. Of these, a histidine residue serves to activate the hydroxyl nucleophile and a lysine assists in stabilizing the PAP leaving group. Interestingly, a conserved serine seems to be involved in modulating the activity of these enzymes to prevent PAPS hydrolysis in the absence of acceptor substrate.

Phosphorylation of sugar residues also occurs (e.g., at C-2 of xylose in proteoglycans), but little is known about the nature of this reaction. Phosphoglycosylation, a process in which a sugar phosphate is transferred from a nucleotide sugar donor directly to a serine residue on a protein (e.g., GlcNAc-P-serine), occurs in Dictyostelium. Mannose-6-phosphate on the N-glycans of lysosomal enzymes serves as a targeting signal. However, the formation of this phosphate ester is not mediated by a typical ATP-using kinase, but rather by a two-step reaction involving the donor UDP-GlcNAc (see Chapter 30). Detailed studies of reaction mechanisms have not yet been undertaken.

O-Acetylation occurs in bacteria and in the modification of sialic acids, but little information is available about the chemistry of these reactions. N-Deacetylation of N-acetylglu-cosamine residues occurs during heparin/heparan sulfate formation (see Chapter 16), lipopolysaccharide assembly (see Chapter 20), and GPI-anchor synthesis (see Chapter 11). The bacterial enzyme is zinc dependent, but in-depth studies of the vertebrate N-deacetylases have not been performed. N-Deacetylation of N-acetylneuraminic acid (the most common sialic acid) has also been reported (see Chapter 14).

Figure 5.2

FURTHER READING