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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. doi: 10.1101/glycobiology.3e.005

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Essentials of Glycobiology [Internet]. 3rd edition.

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Chapter 5Glycosylation Precursors

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Published online: 2017.

In nature, most glycans are synthesized by glycosyltransferases, enzymes that transfer activated forms of monosaccharides from nucleotide sugars and lipid-linked sugar intermediates to acceptors including proteins, lipids, and growing glycan chains. Monosaccharide precursors are imported into the cell, salvaged from degraded glycans, or created enzymatically from other sugars within the cell. In eukaryotic cells glycosylation occurs mostly in the Golgi apparatus, even though monosaccharide activation and interconversions occur mostly in the cytoplasm. Nucleotide sugar–specific transporters carry activated sugar donors into the Golgi. In some cases, nucleotide sugars are used to synthesize activated lipid-linked intermediates before glycan transfer. This chapter describes how cells accomplish these tasks, with an emphasis on animal cells.


Glucose and fructose are the major carbon and energy sources for organisms as diverse as yeast and humans. Most organisms can synthesize the other monosaccharides needed for glycan biosynthesis from these sources. Not all of these biosynthetic pathways are equally active in all types of cells. However, there are some general principles. Monosaccharides must be activated to a high-energy donor for use in glycan synthesis. This process requires nucleoside triphosphates (such as UTP or GTP) and a glycosyl-1-P (monosaccharide with a phosphate at the anomeric carbon). They can be activated by a kinase (reaction 1) or generated from a previously synthesized activated nucleotide sugar (reactions 2 and 3):

Image ch5uf01.jpg

The most common nucleotide sugar donors in animal cells are shown in Table 5.1. Sialic acids and their evolutionary ancestors (prokaryotic nonulosonic acids and Kdo) are the only monosaccharides in animals activated as CMP-mononucleotides. Iduronic acid does not have a nucleotide sugar parent because it is formed by epimerization of glucuronic acid after it is incorporated into glycosaminoglycan (GAG) chains. In some instances, one nucleotide sugar can be formed from another either by direct epimerization (reaction 2 above) or by a nucleotide exchange reaction (reaction 3 above). For example, UDP-Gal is made from UDP-Glc by exchange of Gal-1-P for Glc-1-P.

TABLE 5.1.

TABLE 5.1.

Activated sugar donors in animal cells


Three types of sugar transporters carry sugars across the plasma membrane into cells. First are energy-independent facilitated diffusion transporters such as the glucose transporter (GLUT) family of hexose transporters found in yeast and most mammalian cells. The genes encoding these proteins are named SLC2A (solute carriers 2A). Second are energy-dependent transporters—for example, the sodium-dependent glucose transporters (SGLT; gene names SLC5A) in intestinal and kidney epithelial cells. The third type includes transporters that couple ATP-dependent phosphorylation with sugar import. These are found in bacteria (Chapter 21) and are not covered in this chapter.

GLUT family transporters were first described in yeast, where at least 18 genes are now known. Humans have 14 GLUT homologs. GLUT transporters range in size from (∼40–70 kDa) and have similar structures containing 12 membrane-spanning domains, which is typical of many eukaryotic transporters. The transmembrane domains form a barrel with a small pore for sugar passage. Compared with GLUT1, the other family members have a modest 28%–65% amino acid identity. There are “sugar transporter signatures” consisting of one or a few amino acids in specific positions relative to the membrane-spanning domains, but no major transporter motifs.

Typically, the GLUTs have Km values for glucose uptake in the 1–20 mm range. In yeast, many transport glucose, but others are specific for galactose, fructose, or disaccharides. Most mammalian GLUT proteins transport glucose or fructose with variable efficiency, but without fully characterized specificity. However, GLUT5 primarily transports fructose, and the GLUT called HMIT is a proton-coupled myo-inositol transporter. GLUT2 also efficiently transports glucosamine.

Glucose is transported from the gut lumen by an energy-requiring Na+-dependent glucose transporter (SGLT-1) and is recovered from the kidney filtrates by a related transporter (SGLT-2). The SGLT-type transporters have Km values of <1 mm for glucose.

GLUT1–5 have different distributions among different mammalian cells and different Km values that enable them to respond to the availability of glucose. Although most of the human GLUT members are located on the cell surface, a portion of GLUT4 resides in intracellular vesicles, which are recruited to the cell surface in response to insulin. Following carbohydrate-rich meals, glucose transported by SGLT-1 in the intestine is thought to promote the recruitment of GLUT2 to the apical surface for enhanced glucose uptake.



Monosaccharides can also be salvaged from glycans degraded within cells (Chapter 44). Most of the degradation occurs at low pH in lysosomes. Salvage pathways have received relatively little attention, but their contribution to glycosylation may be quite substantial. For example, 80% of the radiolabeled N-acetylglucosamine from glycoproteins degraded in liver lysosomes is converted into UDP-GlcNAc and at least one-third is used to synthesize secreted glycoproteins. Also, fibroblasts endocytose labeled glycans and reuse about 50% of the amino sugars for new glycoprotein synthesis. Efficient salvage is not limited to GlcNAc. Much of the sialic acid derived from endocytosed extracellular glycans may be reused for new glycoprotein synthesis.

Monosaccharides released by degradation must exit the lysosome. Different lysosomal carriers exist for neutral hexoses (glucose, mannose, and galactose), N-acetylated amino sugars, and acidic sugars; the neutral sugar carrier has a Km value of 50–75 mm for hexose substrates, but also transports fucose and xylose. The N-acetylhexosamine carrier (Km ∼ 4 mm) cannot transport nonacetylated amino sugars. The sialic acid and glucuronic acid carrier (Km ∼ 300–550 µm) is important because its loss leads to an accumulation of these sugars in the lysosome and secretion into the urine, with genetic mutations resulting in a human lysosomal storage disease (Chapter 44). Most monosaccharides that reach the cytoplasm are activated and reused, as described below. However, the uronic acids cannot be reused in animals and are degraded via the pentose phosphate pathway. Mannose released from N-glycan processing or turnover is transported out of the cell by a hexose transporter/exchanger with little or no direct reutilization.

Activation and Interconversion of Monosaccharides


Glycogen is an immense molecule that contains up to 100,000 glucose units, arranged in Glcα1–4Glc repeating disaccharides with periodic α1–6Glc branches. It is synthesized on a cytoplasmic protein called glycogenin (Chapter 18). Glycogen is the major storage polysaccharide in animal cells, and its synthesis and degradation (glycogenolysis) are highly regulated for energy use. Glycogen is synthesized by the addition of single glucose units from UDP-Glc, and it is degraded by glycogen phosphorylase. This non-ATP-dependent reaction forms glucose-1-P by phosphorolysis of glycogen. This substrate can be used directly to form UDP-Glc or converted to glucose-6-P for further catabolism via glycolysis or direct oxidation via glucose-6-phosphate dehydrogenase.


Glucose is the central monosaccharide in carbohydrate metabolism, and it can be converted into all other sugars (Figure 5.1). Glucose is first converted to glucose-6-P by hexokinase. In the glycolytic pathway, glucose-6-P is converted to fructose-6-P by phosphoglucose isomerase or into glucose-1-P by phosphoglucomutase. Reaction of glucose-1-P with UTP forms the high-energy donor UDP-Glc. The UDP-Glc pool is quite large, and it is used to synthesize glycogen and other glucose-containing molecules such as glucosylceramide (Chapter 11) and dolichol-P-glucose, which is used in the N-linked glycan biosynthetic pathway (Chapter 9).

FIGURE 5.1.. Biosynthesis and interconversion of monosaccharides.


Biosynthesis and interconversion of monosaccharides. The relative contributions of each pathway under physiological conditions are unknown. (Rectangles) donors; (ovals) monosaccharides; (asterisks) control points; (KDN) 2-keto-3-deoxy-D-glycero-D-galactonononic (more...)

Some glucose-6-phosphate is the substrate for glucose-6-P dehydrogenase, the entry point for the oxidation via the pentose phosphate pathway that subsequently produces 6-phosphogluconate and then ribose-5-phosphate. These reactions generate nicotinamide adenine dinucleotide phosphate (NADPH), which is needed to maintain proper redox status.

Glucuronic Acid

UDP-GlcA (glucuronic acid) is synthesized directly from UDP-Glc by a two-stage reaction requiring two NAD+-dependent oxidations at C-6. UDP-GlcA is used primarily for GAG biosynthesis (Chapters 16 and 17), but some N- and O-linked glycans and glycosphingolipids contain glucuronic acid as well. The addition of glucuronic acid to bile acids and xenobiotic compounds (such as drugs and toxins) increases their solubility, and a large class of glucuronosyltransferases is devoted to these reactions.

Iduronic Acid

Iduronic acid is the C-5 epimer of GlcA, and it is found in GAGs dermatan sulfate, heparan sulfate, and heparin. Unlike all other monosaccharides found in glycans, iduronic acid is not directly synthesized from a nucleotide sugar donor. Instead, it is generated by epimerization of GlcA following its incorporation into the growing GAG chain (Chapter 17).


Decarboxylation of UDP-GlcA gives UDP-Xyl, which is used to initiate GAG synthesis (Figure 5.2; Chapter 17) in vertebrates. Xylose is also found on proteins that have O-glucose modifications in epidermal growth factor (EGF) modules (Chapter 13) and on O-mannose-based glycans on α-dystroglycan (Chapters 13 and 45), as well as on plant N-glycans. A type II membrane protein performs the decarboxylation reaction using UDP-GlcA transported into the endoplasmic reticulum (ER) or Golgi. In Caenorhabditis elegans (C. elegans), the decarboxylase is called SQV-1, and it colocalizes with the UDP-GlcA transporter (Chapter 25). In Arabidopsis, another UDP-GlcA decarboxylase also occurs in the cytoplasm, but no ortholog has been identified in animals.

FIGURE 5.2.. Biosynthesis of UDP-xylose and the branched sugar donor UDP-apiose from UDP-GlcA.


Biosynthesis of UDP-xylose and the branched sugar donor UDP-apiose from UDP-GlcA. Xylose is found in animals and plants, whereas apiose is used for plant polysaccharides such as apiogalacturonan in Lemna minor. Note the similarity and overlap in the synthesis (more...)


Mannose is used for multiple types of glycans (Chapters 9, 11, 12, and 13). Guanosine diphosphate mannose (GDP-Man) is the primary activated donor. Its production requires prior synthesis of mannose-6-P and its conversion to mannose-1-P. Two ways to produce mannose-6-P are by direct phosphorylation via hexokinase or conversion of fructose-6-P to mannose-6-P using the enzyme phosphomannose isomerase. In yeast, loss of the latter enzyme is lethal. In humans, loss of this enzyme produces a potentially fatal disease called congenital disorder of glycosylation (CDG-type Ib or MPI-CDG) (Chapter 45). Phosphomannose isomerase is important because free exogenous mannose is not common in the diet, and this enzyme is the key link between mannose and glucose. Both yeast and human phosphomannose isomerase deficiencies can be rescued by providing exogenous mannose, but too much mannose is toxic. Mice totally lacking phosphomannose isomerase activity die in utero because of an accumulation of Man-6-P that inhibits glycolysis and depletes ATP. In mammals, mannose-6-P is converted to mannose-1-P using phosphomannomutase. Because mannose-6-P and mannose-1-P are both obligate precursors of GDP-Man, failure to make sufficient amounts of either one reduces the formation of GDP-Man, which is a direct donor for lipid-linked oligosaccharides (see below) and a precursor for dolichol-P-mannose, which serves multiple glycosylation pathways.

Mannose-6-P can also condense with phosphoenolpyruvate to form 2-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN). This molecule is activated with CTP to produce CMP-KDN, and is abundant in fish (e.g., in trout testis and on their sperm), where it is thought to be important for sperm–egg adhesion.


Guanosine diphosphate fucose (GDP-Fuc) can be derived from GDP-Man by the sequential action of two enzymes involving three steps. In the first step, the C-4 hydroxyl group of GDP-Man is oxidized to a ketone (GDP-4-keto-6-deoxy-mannose) by the enzyme GDP-Man 4,6-dehydratase along with the reduction of NADP+ to NADPH. The next two reactions are catalyzed by a single polypeptide that has epimerase and reductase activity and is well conserved from bacteria to mammals. GDP-4-keto-6-deoxymannose is epimerized at C-3 and C-5 to form GDP-4-keto-6-deoxyglucose, which is then reduced with NADPH at C-4 to form GDP-Fuc (Figure 5.3A). The first dehydration step is feedback inhibited by GDP-Fuc. GDP-Fuc can also be synthesized directly from fucose. The first step uses a kinase to make fucose-1-P, which is then converted to GDP-Fuc. Mutant CHO cells that cannot convert GDP-Man to GDP-Fuc form hypofucosylated proteins, but this can be corrected by providing exogenous fucose in the medium. Also, mice genetically deficient in the GDP-Man to GDP-Fuc conversion can be rescued by providing fucose in their food or drinking water. Plasma membrane transporters for sugars other than glucose have been characterized and fucose transporters may exist as well, although they have not been fully characterized, so their quantitative contribution is not known. As with many monosaccharides other than glucose, free fucose concentration in the blood is in the very low micromolar range.

FIGURE 5.3.. Conversion of activated sugar donors.


Conversion of activated sugar donors. Steps in the synthesis of (A) GDP-Fuc from GDP-Man and (B) UDP-Gal from UDP-Glc. Details of the various enzymes are given in the text. GDP-Fuc synthesis by this route is not reversible, whereas the interconversion (more...)


Activated uridine diphosphate galactose (UDP-Gal) can be made by direct phosphorylation at C-1 to give galactose-1-P, which reacts with UTP to form UDP-Gal. Alternatively, galactose-1-P can be converted to UDP-Gal via a uridyl transferase exchange reaction with UDP-Glc that displaces glucose-1-P. A deficiency in this activity results in a severe human disease called galactosemia, which leads to intellectual disability, liver damage, and eventual death if galactose intake is not controlled (Chapter 45). Finally, UDP-Gal can be formed from UDP-Glc by the NAD-dependent reaction catalyzed by UDP-Gal 4-epimerase. The enzyme first converts the C-4 hydroxyl group to a keto derivative forming NADH from bound NAD+. In the next step, the keto group is converted back to a hydroxyl group with opposite orientation and NAD+ reforms (Figure 5.3B). The same enzyme interconverts UDP-GalNAc and UDP-GlcNAc.

Galactose usually occurs as a pyranose (p) ring in “higher” animals, but bacteria and pathogenic eukaryotes such as Leishmania and Aspergillus incorporate galactofuranose (f) into their glycans (Chapter 21). The donor is formed by conversion of UDP-Gal(p)→UDP-Gal(f) using a flavin adenine dinucleotide–dependent mutase.


Synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) begins with the formation of glucosamine-6-P from fructose-6-P by transamination using glutamine as the –NH2 donor. Glucosamine-6-P is then N-acetylated via an acetyl-CoA-mediated reaction to form N-acetylglucosamine-6-P and then isomerized to N-acetylglucosamine-1-P via a 1,6-bis-phosphate intermediate. Similar to the other activation reactions, N-acetylglucosamine-1-P then reacts with UTP to form UDP-GlcNAc and pyrophosphate. Alternatively, GlcNAc can be directly phosphorylated to form N-acetylglucosamine-6-P by a kinase that also synthesizes N-acetyl-mannosamine-6-P from N-acetylmannosamine. Phospho-N-acetylglucosamine mutase then converts N-acetylglucosamine-6-P to N-acetylglucosamine-1-P. This route may account for the efficient salvage of GlcNAc from lysosomal degradation of glycans. Glucosamine can also be salvaged following sequential phosphorylation and acetylation.


Uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) can arise from two routes. One is the direct reaction of N-acetylgalactosamine-1-P with UTP. N-Acetylgalactosamine-1-P is formed by a specific kinase that is distinct from galactose-1-kinase. UDP-GalNAc can also be formed by epimerization of UDP-GlcNAc using the same NAD-dependent epimerase that converts UDP-Glc to UDP-Gal.

Sialic Acids

The term sialic acid is the name given to a group of more than 50 different variations of the three parent compounds, N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and KDN (mentioned above) as discussed more fully in Chapter 15. Generation of cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) is more complicated than formation of the other activated sugars. First, UDP-GlcNAc is converted to N-acetylmannosamine-6-P by a single enzyme with two catalytic activities. The first activity epimerizes the GlcNAc of UDP-GlcNAc at C-2 and cleaves the UDP to yield N-acetylmannosamine (ManNAc). In the next reaction, the kinase activity uses ATP to form ManNAc-6-P. Mutations in this enzyme cause two completely distinct metabolic disorders: sialuria and inclusion body myopathy type 2 (Chapter 45). Knocking out this gene in mice causes early embryonic lethality. In the next step, ManNAc-6-P is condensed with phosphoenolpyruvate to form N-acetylneuraminic acid-9-P. Phosphate is then removed by a phosphatase. Activation with CTP yields CMP-Neu5Ac, which is the target for a hydroxylase that converts some of it to CMP-Neu5Gc. The last steps occur in the nucleus with subsequent export of the activated precursors to the cytoplasm. Other modifications of sialic acid can occur in the Golgi after transfer of the sialic acid to the oligosaccharide acceptor.

Sialic acids can be salvaged from internal glycoprotein turnover or from plasma and activated by phosphorylation and the addition of CMP from CTP. In addition, GlcNAc can be activated to UDP-GlcNAc and reenter the biosynthetic pathway to CMP-Neu5Ac (Chapter 15).

Diverse Monosaccharides in Bacteria and Plants

Fucose is the only deoxyhexose commonly found in animal cell glycans. In contrast, bacterial and plant polysaccharides and glycoproteins frequently contain a variety of deoxysugars, deoxyaminosugars, and branched-chain sugars. These diverse sugars often have potent biological properties. For example, aminoglycoside antibiotics like streptomycin bind to the bacterial ribosome to disrupt protein synthesis. Deoxyhexoses are often immunological determinants of lipopolysaccharides or O-antigens of the Salmonella species. Five of the eight possible 3,6-dideoxyhexoses have been found in these organisms at the nonreducing end of the Gram-negative cell wall lipopolysaccharide. Other deoxyhexoses, such as a 4,6-dideoxy-hexose and a 2,3,6-trideoxyhexose, are also biologically significant but so far seem to be uncommon in nature.

Biosynthesis of both deoxysugars and dideoxysugars begins with the oxidation of C-4, similar to the first step of the conversion of GDP-Man to GDP-Fuc. The nucleotide (N) differs for the various sugars, and the individual pathways use different dehydratases. For example, biosynthesis of most 3,6-dideoxyhexoses (except colitose) begins with conversion of CDP-glucose to CDP-4-keto-6-deoxyhexose by NAD+-dependent CDP-glucose dehydratase. In the biosynthesis of abequose (3,6-dideoxy-D-xylohexose), the product, CDP-6-deoxy-L-threo-D-glycero-hexulose, is then converted in two additional steps to CDP-3,6-dideoxy-D-glycero-D-glycero-4-hexulose by a second dehydratase followed by a reductase.

Amino sugars, such as glucosamine, arise from keto sugars by the addition of an amino group from glutamine (Figure 5.1). In addition, bacteria and plants have many 6-deoxy-hexoses with amino groups in the 2, 3, or 4 positions. For example, duanosamine is a 3-amino-6-deoxyhexose that is found in the antibiotic duanomycin. Here, TDP-glucose is dehydrated to 3-keto-6-deoxyglucose and the amino group is added via a transamination reaction probably involving a vitamin B6-dependent reaction.

Plants and bacteria also contain a number of branched-chain sugars. For instance, apiose is a component of the polysaccharide apiogalacturonan of Lemna minor, and strepose is a component of the antibiotic streptomycin produced by Streptomyces griseus. Apiose (Figure 5.2) is synthesized from UDP-GlcA via a 4-keto intermediate that can yield UDP-Xyl or UDP-apiose. Apiose synthesis removes carbon 3 from the chain to give the branched sugar by an unknown mechanism. Although the synthesis of other branched-chain sugars has not been delineated, they probably follow similar reaction pathways.


In eukaryotes, nucleotide sugars are synthesized in the cytoplasm or nucleus, whereas most glycosylation occurs inside the ER or Golgi compartments (exceptions being hyaluronan [Chapter 16] and nucleocytoplasmic glycosylation [Chapters 18 and 19]). Therefore, newly synthesized nucleotide sugars are on the “wrong” side of the membrane for most glycosylation reactions, and so must be transported into the ER and Golgi. Negative charge prevents these donors from simply diffusing into these compartments. To overcome this hurdle, eukaryotic cells have a set of energy-independent nucleotide sugar antiporters that deliver nucleotide sugars into the lumen of these organelles with the simultaneous exiting of nucleoside monophosphates; most of which must first be generated from the nucleoside diphosphates by a nucleoside diphosphatase (Figure 5.4). This transport mechanism was determined biochemically in isolated vesicles and genetically in various mutant cell lines. The Km of the transporters ranges from 1 to 10 µm. Using in vitro systems, the transporters have been shown to increase the concentration of the nucleotide sugars within the Golgi lumen by 10- to 50-fold. This is usually sufficient to reach or exceed the calculated Km of glycosyltransferases that use these donors.

FIGURE 5.4.. Some known transporters for nucleotide sugars, PAPS (3′-phosphoadenosine-5′-phosphosulfate), and ATP are located in the Golgi membranes of mammals, yeast, protozoa, and plants.


Some known transporters for nucleotide sugars, PAPS (3′-phosphoadenosine-5′-phosphosulfate), and ATP are located in the Golgi membranes of mammals, yeast, protozoa, and plants. These proteins are antiporters and return the corresponding (more...)

Most of these antiporters are found in the Golgi, but some are also found in the ER. They are organelle-specific and their location usually corresponds to the location of the relevant glycosyltransferases (Table 5.2 and Figure 5.4). Nucleotide sugar imported into the Golgi is not energy-dependent or affected by ionophores. However, the import is competitively inhibited by the corresponding nucleoside monophosphates and diphosphates in the cytosol, but not by the monosaccharides. There are also transporters for ATP and PAPS (3′-phosphoadenosine-5′-phosphosulfate), which are used for carbohydrate and protein sulfation.

TABLE 5.2.

TABLE 5.2.

Nucleotide transport in animal cell Golgi and ER

The glucuronidation of bile and xenobiotic compounds in the ER explains the need for UDP-GlcA transporter in the ER. The presence of a Golgi transporter is consistent with the location of the glycosyltransferases that use UDP-GlcA for the formation of GAG chains and other classes of glycans. The observation that reglycosylation of misfolded glycoproteins occurs in the ER (Chapter 39) explains the need for an ER UDP-Glc transporter. Under stressful conditions that activate the unfolded protein response, synthesis of lumenal uridine diphosphatase increases to accommodate increased transport of UDP-Glc needed for reglucosylation of misfolded glycoproteins. The existence of UDP-GlcNAc, UDP-GalNAc, and UDP-Xyl transporters in the ER may mean that some reactions thought to occur exclusively in the Golgi may also occur in the ER. A good example is the synthesis of O-fucosylated proteins such as Notch in the ER versus fucosylation of N- and O-linked chains in the Golgi (Chapter 13). Other, as-yet-undiscovered, glycosylation reactions may also occur in the ER.

Mutations in several nucleotide sugar transporters, UDP-Gal, CMP-Sia, GDP-Fuc, UDP-GlcA/UDP-GalNAc, and UDP-GlcNAc cause human glycosylation disorders (Chapter 45) generating incomplete sugar chains. Mutant mammalian cell lines also lack specific nucleotide sugar transporters (e.g., for UDP-Gal or CMP-Sia; Chapter 49). However, there is some “leakiness” in such mutants. For instance, loss of the UDP-Gal transporter in the Golgi of mutant MDCK (Madin–Darby canine kidney) cells decreases the synthesis of keratan sulfate and galactosylated glycoproteins and glycolipids, but leaves heparan and chondroitin sulfate unaffected. This is probably because the galactosyltransferases that synthesize the core region tetrasaccharide common to GAG chains have lower Km values for their donors (Chapter 17).

Many putative transporters were identified by homology in the genomes of mammals, Drosophila melanogaster (D. melanogaster), C. elegans, plants, and yeast. Like the plasma membrane GLUT transporters discussed above, all are multimembrane-spanning (type III) proteins, but the level of amino acid identity does not give any clue to the substrate specificity. The UDP-GlcNAc transporters from mammalian cells and yeast are 22% identical, whereas mammalian CMP-Sia, UDP-Gal, and UDP-GlcNAc transporters have 40%–50% identity. Clever domain-swapping experiments show that distinct regions are responsible for functional transport, and engineered chimeric transporters can carry both CMP-Sia and UDP-Gal.

Heterologous expression or rescue of transporter-deficient cell lines can be used to analyze the function of the putative transporters. For example, expressing the C. elegans gene SQV-7 in yeast showed that this one protein transports UDP-GlcA, UDP-GalNAc, and UDP-Gal, whereas mutant alleles cannot transport any of these donors. The human gene SLC35B4 encodes a bifunctional transporter that recognizes UDP-Xyl and UDP-GlcNAc. Another example is the GDP-Man transporter of Leishmania that can also transport GDP-Fuc and GDP-arabinose. This point illustrates that functional, biochemical analyses are essential; genetic homology is insufficient to infer specificity. Moreover, not all of the potential transport-like genes have been assigned a specific substrate.

Theoretically, glycosylation may be controlled in part by regulating the availability of nucleotide sugars within the Golgi, presumably by regulating the transporters. The subcompartmental location (cis, medial, trans) of the transporters in the Golgi is not known nor are the physical relationships of the transporters to the various glycosyltransferases they service. Clearly, a functional Golgi compartment requires both the nucleotide sugar donor and the acceptor with a colocalized transferase. There have been few studies on how the actual glycosylation reactions occur within the Golgi. Is it more like solution chemistry or like solid-state transfers? Are there really “soluble pools” of nucleotide sugars? Dramatic time-lapse videos of green fluorescent protein (GFP)-tagged glycosyltransferases show that the proteins are highly mobile within the Golgi, but there is also physical evidence for multiglycosyltransferase complexes involved in the biosynthesis of N-linked glycans, glycosphingolipids, and heparan sulfate. Many transporters appear to function as homodimers, and the GDP-Man transporter in Saccharomyces cerevisiae (S. cerevisiae) (VRG4) oligomerizes in the ER and appears to be transported to the Golgi by an active process. Also, synthesis of galactosylceramide occurs in the ER and a portion of the UDP-Gal transporter binds specifically to galactosylceramide transferase and is retained in the ER to provide donor substrate (Chapter 11).


Biochemical control of the intracellular concentrations of glycosylation precursors (ultimately nucleotide sugars) is a complex and important area of ongoing research. In some cases, key biosynthetic enzymes are inhibited by their final products. A human genetic disorder called sialuria is a clear example. In this condition, massive amounts of sialic acid (several grams each day) are secreted into the urine along with various intermediates in the CMP-Sia biosynthetic pathway. Sialuria is caused by mutations in the enzyme responsible for the first step in sialic acid biosynthesis, N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE). Sialuria-causing mutations impair the normal feedback inhibition of GNE by CMP-Sia, the end product of the precursor pathway (Chapter 45).

Most of the precursor pools turn over within a matter of minutes. The relevant steady state concentrations of the nucleotide sugars have been difficult to reliably determine. It is especially difficult to measure the concentrations of nucleotide sugar donors at their sites of action, which are often confined to the lumen of the ER or Golgi. In whole-animal studies, the GDP-Fuc pool and fucosylated glycans in the intestine can be regulated by the diet and time of weaning. Considering that resident bacteria in the small intestine participate in the induction of fucosylation pathways in the enterocytes, and that gut bacteria can release and use monosaccharides, dietary manipulation of glycosylation introduces another level of unexplored complexity. The relationship of amino acid and nucleotide metabolism to nucleotide sugar metabolism is also potentially important but largely unexplored.


Glycans can be modified, imparting additional complexity and biological information. Sulfation, phosphorylation, methylation, pyruvylation, acetylation, and acylation have been found and their donors are listed in Table 5.3. In “lower” eukaryotes and bacteria, pyruvic acid is often found as a 1-carboxyethylidene bridge between two hydroxyl groups on a sugar such as galactose. Because all of these reactions occur in the Golgi in eukaryotes, there must be carriers or transporters that deliver and orient activated donors for efficient synthesis. As additional modifications of sugar chains made in the ER–Golgi pathway are uncovered, they will likely turn out to require specific transporters to carry the activated donors into the lumen of these compartments.

TABLE 5.3.

TABLE 5.3.

Donors for glycan modifications


Multiple glycosylation pathways in prokaryotes and eukaryotes require lipid carriers to present monosaccharides and oligosaccharides at the proper location. Undecaprenyl-P (bactoprenol) is the glycosyl carrier for O-antigen, peptidoglycan, capsular polysaccharides, teichoic acid, and mannans in bacteria (Chapter 21). Dolichol-P serves the same function in eukaryotic cells (Chapter 9). Dolichol-P-mannose provides all of the mannose for glycophospholipid anchors, C-mannosylated proteins, O-mannose-based chains, and four of the mannose residues of the precursor oligosaccharide used for N-glycan biosynthesis. Dolichol-P-glucose provides glucose for the mature N-linked glycan precursor Glc3Man9GlcNAc2, which itself is built on dolichol pyrophosphate (dolichol-PP).

The formation of dolichol-P involves elongation of farnesyl pyrophosphate with multiple cis-isopentenyl pyrophosphate units. The total number of isoprene units can vary from typically 11 in bacteria (making a C55 bactoprenol chain) up to 21 in mammals. In eukaryotes, the double bond nearest the pyrophosphate must be reduced for the carrier to be functional in glycosylation. Studies in yeast, mice, and humans indicate that direct reduction of polyprenol to dolichol is a major pathway, but an alternate pathway must also exist. It is unclear whether the phosphates are removed before or after the reduction step. The evolutionary significance of the different chain lengths and reduction of the double bond is not known. Dolichol is phosphorylated by an ATP-dependent dolichol kinase to generate dolichol-P as needed. Because dolichol, dolichol-P, and dolichol-PP are all generated from a common metabolically stable pool, they must be recycled and interconverted as needed. Dolichol occurs in the ER and Golgi and turns over very slowly.


The authors acknowledge contributions to previous versions of this chapter by the late Alan Elbein and appreciate helpful comments and suggestions from Vivek Kumar, Simone Kurz, and Jeremy Praissman.


Copyright 2015-2017 by The Consortium of Glycobiology Editors, La Jolla, California. All rights reserved.

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Bookshelf ID: NBK453043PMID: 28876856DOI: 10.1101/glycobiology.3e.005


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