Glycan Biosynthesis in Mammals - CHEMICAL BIOLOGY

CHEMICAL BIOLOGY

Glycan Biosynthesis in Mammals

  1. Adam Meledeo and Kevin J. Yarema, Department of Biomedical

Engineering, The Johns Hopkins University, Baltimore, Maryland

doi: 10.1002/9780470048672.wecb190

Mammalian glycosylation, the process by which a cellular complement of glycans of dazzling complexity is produced, involves a minimum of 1% of the genome (in humans) and is the dominant postsynthetic modification of both proteins and lipids endowing these molecules with an extended range of structure and function. This article provides a brief outline of the metabolic process by which monosaccharides—the raw material for the construction of complex oligosaccharides—are converted into high energy nucleotide sugar ''building blocks'' that are in turn assembled into four major classes of mammalians carbohydrates: glycoproteins, glycolipids, GPI anchored structures, and polysaccharides. Finally, the article concludes with a brief discussion of modern methods for manipulating glycans in living cells and in animals-using synthetic, molecular biology, and ''chemical biology'' approaches—as early steps toward developing sugar-based medicines.

As more information is uncovered regarding the human genome and proteome, it becomes increasingly clear that a deeper level of information resides in the great variety of signaling, receptor, and structural molecules that comprise the human body than is predicted from a strict examination of the genetic code. The answer to this apparent disparity lies in the posttranslational modification of proteins that endows the unexpectedly modest number of genes with great product diversity. Although dozens of posttranslational modifications occur, glycosylation—the addition of one or more sugar residues to a protein or lipid to convey additional information, structure, or function—is arguably the most common (1). In recent years, major strides in the development of sensitive and reliable methods of detection and functional analysis for complex carbohydrates have revealed just how significant these posttranslational glycosylation modifications are; yet, many of these structures remain elusive as large-scale sugar-specific technologies have lagged the rapid rate of discovery for genes and proteins. Now, information gleaned from glycomics is accelerating the study of glycosylation (2, 3) and has generated claims that 220-250 genes, roughly 1% of the human genome, are involved in glycan production and modification (4), and that a minimum of 50% of proteins are glycosylated (5). This article outlines the biosynthetic process of mammalian glycans by first examining the basic monosaccharide building blocks and the ways they combine to form oligosaccharide and polysaccharide structures (Fig. 1). An inspection of the various glycoprotein, glycolipid, GPI anchor, and independently functional polysaccharide linkages is then provided along with a brief description of the structure and function of the various classes of enzymes in the respective biochemical pathways. The article concludes with examples of current research efforts in synthetic chemistry, biologic, and “chemical biology” strategies that seek to exploit the flurry of recent advances in understanding mammalian glycans to develop novel sugar-based therapies for human disease.

Figure 1. Overview of mammalian glycan biosynthesis. Monosaccharides, most commonly glucose (shown), are imported into a cell by membrane transporters of the GLUT or SGLT families. Imported sugars can undergo extensive processing in the cytosol, as exemplified by ManNAc conversion to sialic acid (see Fig. 7), or be directly phosphorylated and converted to high energy nucleotide sugars (an NDP-monosaccharide is shown, where N is a nucleoside). Nucleotide sugars may be used in the cytosol to synthesize lipid-linked precursors of N-linked glycans (see Fig. 3), or they may be transported into the lumen of the ER or Golgi by nucleotide sugar transporters (NSTs) and therein used as donors for oligosaccharide biosynthesis catalyzed by glycosyltransferases (GTs). The NDP released by GT action is converted to NMP by pyrophosphatase (PPase). Upon further processing and transport, glycans are displayed on the cell surface (shown) or exported from the cell (not shown) where they have diverse functions ranging from signaling and metabolic regulation (e.g., glycoprotein hormones) to a structural role (e.g., polysaccharides that are major ECM constituents).

Monosaccharides—The Building Blocks for Glycosylation

Monosaccharides are obtained from the diet and transported into cells

In mammals, the primary source for the monosaccharides used for glycan biosynthesis is the diet, but many cell types also scavenge sugars released into the bloodstream by other tissues and organs. A typical mammalian diet containing polysaccharides and starches will result in a rich supply of simple sugars such as glucose (Glc) after digestion in the gastrointestinal tract (the full names, abbreviations, and chemical structures of mammalian monosaccharides are given in Fig. 2). These sugars, as well as several less abundant monosaccharides such as galactose (Gal), mannose (Man), or glucosamine (GlcN), are absorbed into the bloodstream and taken up by cells throughout the body via transporters located in the plasma membrane.

Characterization of the function and structure of monosaccharide transporters found in the plasma membrane led to the identification of the SGLT (sodium-dependent co-transporters from the gene SLC5A) and GLUT (sodium-independent facilitative transporters from the gene SLC2A) families (6, 7). Various members of these two transporter families are localized to different tissue types. For example, GLUT1 is found in erythrocytes; GLUT4, GLUT5, and GLUT12 predominate in skeletal muscle tissue although other transporters are also expressed at lower numbers in this metabolically voracious tissue (8); and GLUT14 is specifically expressed as the predominant transporter in two alternative splice forms of the human (but not mouse) testis (9). Another feature of these proteins is overlapping substrate specificity with many family members capable of transporting multiple monosaccharides, albeit with differing efficiencies (10).

Figure 2. Mammalian monosaccharides and nucleotide sugars. (A) Chemical structures, common names, and abbreviations of the 11 monosaccharides found in mammalian glycans as well as ManNAc, the precursor for sialic acids. (B) Examples of the three classes of nucleotide sugars are provided by UDP-GlcNAc (Glc, GlcA, Gal, GalNAc, and Xyl also use UDP), GDP-Man (Fuc also is linked to GDP), and CMP-Neu5Ac/Gc (IdoA is produced by postsynthetic epimerization of GlcA and therefore does not require a nucleotide sugar).

De novo synthesis of high-energy nucleotide sugars

After monosaccharides are delivered into cells, they are subjected to a series of chemical conversions, including epimerization, acetylation, condensation, and phosphorylation reactions, to produce the spectrum of building blocks required for glycan biosynthesis. The intracellular metabolic network is capable of the de novo synthesis of sufficient amounts of glucosamine (GlcN), fructose (Fru), mannose (Man), fucose (Fuc), N-acetylneuraminic acid (sialic acid, Neu5Ac or Sia), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), xylose (Xyl), and glucuronic acid (GlcA), all of which exist in the D-conformation except for L-fucose. Mammals other than humans also produce the N-glycolylneura- minic acid (Neu5Gc) form of sialic acid (11).

In the cytosol, these monosaccharides can be phosphorylated and subsequently coupled with nucleotides such as uridine diphosphate (e.g., UDP-GlcNAc), guanosine diphosphate (e.g., GDP-mannose), or cytosine monophosphate in the case of sialic acids (Fig. 2b) to create a set of high energy “building blocks” for glycan assembly. In some cases, such as for the initial steps in the synthesis of the dolichol-linked 14-mer used in N-linked glycan biosynthesis (discussed below) or for O-GlcNAc protein modification (12), nucleotide sugars are used in the cytosol; more often they are transported into the lumens of the Golgi apparatus and endoplasmic reticulum (ER) where the bulk of oligosaccharide assembly and processing occurs. In either case, the release of the monosaccharide from its bonded nucleotide phosphate provides the energy currency for the formation of glycosidic bonds found in glycolipids and glycoproteins.

Transport of nucleotide sugars into ER/golgi

The transport of high energy nucleotide sugars from the cytosol into the ER and Golgi lumens occurs by highly specific membrane proteins of the SLC35 nucleotide sugar transporter family. This class of proteins has at least 17 members, some of which can accept multiple substrates; at the same time, certain nucleotide sugars can be accepted by multiple transporters. These transporters are organelle specific; typically nucleotide sugars are only transported into an organelle compartment endowed with the corresponding glycosyltransferases (13). For example, CMP-Sia, GDP-Fuc, and UDP-Gal are transported solely into the Golgi; UDP-GalNAc, UDP-GlcNAc, UDP-GlcA, and UDP-Xyl are transported twice as rapidly into vesicles of Golgi as ER; conversely, UDP-Glc is transported into ER vesicles much more rapidly than into the Golgi (10).

Regulation of the assembly of complex carbohydrates from nucleotide sugars, which is an extremely complex and still poorly understood process, is accomplished by several means, including compartmentalization of glycosyltransferases, the activities of these enzymes, nucleotide sugar-transport rates, and the available concentration of substrates (14). By influencing the latter two parameters, nucleotide sugar transporters play a major role in determining the outcome of glycan structure and significant efforts have been expended to understand the transport mechanism. Briefly, these proteins are anti-porters that exchange the nucleotide sugar for a corresponding nucleotide monophosphate in an equimolar fashion (15). For example, a nucleoside monophosphate (NMP) may be exported from the ER in exchange for the importation of a nucleotide sugar (Fig. 1). It is worth noting that NMP is not a product of the glycosylation process within these organelles; instead NMP is produced by enzymatic dephosphorylation of NDP generated during the glycosyltransferase-catalyzed attachment of a monosaccharide residue to a growing oligosaccharide chain. Consequently, both the specific transporter and the corresponding nucleotide diphosphatase are required within the lumen of a specific organelle for successful transport, and evidence suggests that the transport of these nucleotide sugars into the ER or Golgi apparatus regulates which macromolecules will undergo glycosylation (16). Transport is competitively inhibited by corresponding nucleoside monophosphate or diphosphate in the cytosol, but not by the free sugars, and it does not require an energy source such as ATP. Finally, after glycan assembly, the many postsynthetic modifications that glycans undergo (phosphorylation, acetylation, and sulfation) also require active transport of necessary materials into the ER and Golgi; for example, PAPS (3'-phosphoadenosine 5'-phosphosulfate) required for sulfation is imported by the PAPST1 gene product (17).

Glycoconjugate Assembly

Once the required high energy nucleotide sugars and other building blocks have been translocated to the appropriate cellular compartments, the glycosylation of newly synthesized proteins and lipids can begin. In this section we examine the production of the major classes of prevalent mammalian glycan structures; a more thorough discussion, including low abundance glycans not discussed here, can be found in review articles (see the Further Reading list).

Glycoproteins

In proteins there are two major glycan classes, N-linked (Fig. 4) and 0-linked (Fig. 5), based on the atom (nitrogen or oxygen) of the specific amino acid residue to which the glycan is tethered. Glycans are also distinguished from one another by significant differences in biosynthesis, structure, and function.

Figure 3. Outline of N-linked glycoprotein biosynthesis showing topography and major biosynthetic events. Production of the Dol-PP-14-mer begins on the cytosolic side of the ER and is flipped to the luminal side after the assembly of two GlcNAc and five Man residues (a). An additional four Man and three Glc residues are added to create the GlcNAc2Man9Glc3 14-mer that is transferred en bloc by an oligosaccharyltransferase (OST) to a newly synthesized, yet unfolded peptide (b). Trimming of the Glc residues controls protein folding in the calnexin / calreticulin cycle in the ER (c). This is followed by transfer to the c/s-Golgi lumen (d) where removal of four Man residues produces the GlcNAc2Man5 ''core'' structure that is subsequently elaborated into a diverse array of high mannose, complex, and hybrid N-glycans. Enzyme abbreviations are shown (where known), and updated information on enzymatic activity (indicated by the E.C. number) can be found in online databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan Pathway resources (http://www.genome.jp/kegg/glycan/ or http://www.genome.jp/kegg/pathway.html).

Figure 4. Overview of mucin-type O-linked glycoprotein biosynthesis. The production of the eight core structures found in O-glycans is shown along with the enzymes and nucleotide sugars that are involved in each step. Additional information on the enzymes shown can be found in the KEGG databases (see the Legend for Fig. 3).

Figure 5. Overview of glycolipid biosynthesis. Mammalian glycosphingolipids (GSLs) are synthesized from Cer after the addition of Gal, to form the small GalCer-series, or after the addition of Glc and Gal to form the ubiquitous LacCer class, which is subdivided into gangliosides, globosides, and the neo-lactoseries. Additional information on the enzymes and specific GSLs shown can be found in the KEGG databases (see the Legend for Fig. 3).

N-linked glycosylation

N-Linked glycosylation is one of the most prevalent protein modifications and serves many invaluable functions, including stabilization of structure, enhanced solubility, immuno- modulation, mediation of pathogen interaction, serum clearance rate, protein half-life, and proper folding (18). Dysfunctional N-glycosylation can result in serious detriment to the cells and organism as a whole as exemplified by congenital disorders of glycosylation (CDGs) (19) and adult diseases such as cancer (20).

The term “N-linked” refers to the chemical linkage of the glycan moiety to the nitrogen of the amido group of an asparagine (Asn) residue in the host protein. N-linked glycosylation is a multicompartmental affair, which involves the cytosol and both the ER and the Golgi complexes (Fig. 3). Biosynthesis of glycoproteins begins on the cytosolic face of the ER by the formation of a 95-105 carbon polysoprenoid lipid, dolichol phosphate (Dol-P), which acts as a carrier for the nascent glycan structure. Assembly of the core glycan on this carrier begins by addition of a GlcNAc-P (from UDP-GlcNAc) onto Dol-P, forming GlcNAc-pyrophosphoryldolichol (GlcNAc-PP-Dol) through the action of the GlcNAc-1-phosphotransferase DPAGT1. This reaction exemplifies the use of monosaccharide transferases that occurs throughout the assembly of the core structure as well as in subsequent elaboration processes. A second GlcNAc and five Man residues are added (from UDP-GlcNAc and GDP-Man, respectively) in sequence to form Man5GlcNAc2-PP-Dol, which is then flipped to the luminal side of the ER (A in Fig. 3) (21). Additional Man and Glc residues are added in the lumen of the ER via donors Dol-P-Man and Dol-P-Glc, which results in the primary core structure Glc3Man9GlcNAc2-PP-Dol. The terminal α1-2-linked Glc residue is required for recognition by the oligosaccharyltransferase (OST) that attaches this core glycan structure en bloc to the host protein (22).

N -Linked glycosylation is considered to be a cotranslational rather than a posttranslational modification because OST searches unfolded polypeptides emerging from the ER during translation for a universal Asn-X-Ser/Thr consensus sequence, where X is any residue except proline. Proline is disallowed because its rigidity prevents the consensus sequence from forming a loop structure wherein the hydroxyl group of Ser/Thr interacts with the amido group of Asn, thereby making it more nucleophilic and enhancing the installation of the glycan moiety (23). OST binds to Glc3ManpGlcNAc2-PP-Dol and catalytically cleaves the phosphoglycosidic bond in the GlcNAc-P moiety, thereby releasing Dol-PP during the transfer of Glc3Man9GlcNAc2 to the targeted Asn residue (B in Fig. 3) (24).

Once the transfer of the core Glc3Man9GlcNAc2 14-mer to protein is complete, the terminal Glc residues are removed in sequence by glucosidase I (the terminal α1-2-linked Glc) and glucosidase II (the α1-3-linked Glc residue). Glc1Man9GlcNAc2 targets the nascent glycoprotein for entry into the calnexin/ calreticulin cycle within the ER, which is a major component of the quality control system that assists glycoproteins to fold properly and to achieve their ideal conformation (C in Fig. 3) (18, 25). After folding is completed, the final Glc residue is removed by glucosidase II, and a terminal α1-2-linked Man residue is removed from either of the two other arms of the oligosaccharide by ER mannosidase I or II. The remaining Man8GlcNAc2 oligosaccharide structure is transported along with the newly formed protein to the cis-Golgi for further modification (D in Fig. 3). Golgi mannosidases IA and IB subsequently remove three additional α1-2-linked Man residues to form an intermediate Man5GlcNAc2 glycan structure that is subsequently built into the High-Mannose, Complex, and Hybrid subclasses of N-linked glycans.

The formation of complex N-linked glycans begins in the medial-Golgi with the addition of a GlcNAc residue by mannosyl-α1-3-glycoprotein-β1-2-N-acetylglucosaminyltransferase (MGAT1) onto the α1-3-linked Man residue of MansGlcNAc2 (26). Mannosidase II removes the two remaining terminal Man residues from MansGlcNAc2, and mannosyl-α1-6-glycoprotein-β1-2-N-acetylglucosaminyltransferase (MGAT2) adds a GlcNAc residue to the final remaining terminal Man residue (27). Additional modifications, such as the addition of a Fuc to the proximal GlcNAc, addition of a β1-6-GlcNAc to the α1-6-linked Man residue that already bears β1-2-GlcNAc, or capping with terminal sialic acids, generate a wide variety of diverse structures within the complex N-linked glycan class (10). Biosynthesis of hybrid N-linked glycans begins with the addition of β1-2-GlcNAc to the α1-3-linked Man residue of the intermediate Man5GlcNAc2 glycan structure; the removal of the two remaining Man residues by mannosidase II, as occurs in complex N-linked glycan biosynthesis, is prevented by the addition of a β1-4-GlcNAc to the proximal β1-4-Man residue (28). This mannosidase II-protected structure is translocated to the trans-Golgi where additional modifications to the oligosaccharide structure occur, which once again generates a vast array of structures.

O-linked glycosylation

O-Linked glycosylation is a posttranslational modification where the glycan moiety is attached to the hydroxyl group of a serine or threonine amino acid residue of a protein, often in dense clusters of carbohydrate that force the peptide chain into a highly extended, poorly folded conformation. There are several potential O-glycans, including O-linked Fuc and O-glycan linkages to hydroxylysine (in the collagen sequence -Gly-X-Hyl-Gly-) and hydroxyproline (in plants), but by far the most common form of O-glycosylation is the addition of α-N-acetylgalactosamine to form O-linked GalNAc-Ser/Thr (known as mucin-type O-glycosylation) to which many subsequent oligosaccharides can be added for varying functionality. Aberrations in O-linked glycosylation are often found in various disease states, including blood disorders, cancer, and diabetes.

Unlike N-glycosylation that always begins with en bloc transfer of the Glc3Man9GlcNAc2 14-mer core structure, mucin-type O-glycosylation begins in the Golgi apparatus with the addition of a single monosaccharide, typically GalNAc, onto a Ser or Thr residue of a protein that has already completed the folding process (Fig. 4). The production of the initial O-linked GalNAc-Ser/Thr structure, known as the Tn-antigen, is facilitated by an enzyme, O-GalNAc transferase, which forms a complex with the protein. Often, this simple glycan moiety is translocated to the trans-Golgi for elongation through the stepwise addition of Gal, GalNAc, or GlcNAc residues that form the basis of eight core structures. These structures can be further modified by sialylation, sulfation, acetylation, fucosylation, or polylactosamine extension (10).

Although there is not a specifically defined consensus sequence for mucin-type O-linkages (29), statistical analysis has yielded a rule set to predict sites of O-GalNAc modification. For instance, there are inherent differences in site specificity between tissue types based on different GalNAc transferase expression patterns between cells. Moreover, because O-glycosylation occurs on fully folded proteins, only surface-exposed Ser and Thr residues will be accessible for O-glycosylation. The density patterns of O-linked glycans also suggest that nearby residues can influence transferase activity (29).

Also of interest in a discussion of O-linked glycans is the addition of a single GlcNAc to Ser or Thr to form a class of cytosolic and nuclear glycosylated proteins (30). O-GlcNAc is very common in nuclear and cytosolic proteins, including nuclear pore proteins, transcription factors, and cytoskeletal elements (31). O-GlcNAc modification is likened more to phosphorylation than to the other forms of O-glycosylation because of its transient nature and frequent yin-yang status with phosphorylation at the same sites, particularly during different cell-cycle stages and in development (32).

Glycolipids

A glycolipid is any compound containing one or more monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety such as an acylglycerol, a sphingoid, or a prenyl phosphate. In mammals, most glycolipids are glycosphingolipids (GSLs), which is a large and widely varying family of amphipathic lipids based on the ceramide N-acylsphingoid lipid moiety (Fig. 5). GSLs reside in cellular membranes, typically in the plasma membrane where the glycan is almost always oriented outward, exposed to the extracellular space. These molecules play a role in the protective glycocalyx covering of a cell and participate in raft assemblies such as the “glycosynapse” (33). GSLs participate in cell-cell recognition, cell-matrix interactions, and cell surface reception and messaging. GSLs are required for proper development. Biosynthetic or catabolic defects result in pathologies ranging from liver disease to insulin-resistant diabetes, multiple sclerosis, Tay-Sachs, and Graves’ disease.

Glycolipid synthesis begins on the cytosolic face of the ER (34) with the condensation of a serine residue and a palmityl-CoA to form 3-dehydrosphinganine, which is hydroxylated at the 4' oxygen, N-acylated, and unsaturated between C4 and C5 in a trans-fashion to form ceramide (Cer) (35). Ceramide then crosses the ER membrane and undergoes one of several modifications that lead to different classes of glycolipids; most commonly, Cer is conjugated with a Gal or Glc residue to form the simple glycolipids GalCer and GlcCer (Fig. 5). These two glycolipids form the core of all mammalian GSLs; the GalCer core undergoes only a few conservative modifications, whereas the GlcCer core can experience extensive elaborations that generate hundreds of distinct structures.

GalCer-based GSLs are restricted to a few specific cell types, including myelin sheathing provided to neuronal axons by oligodendrocytes and Schwann cells, and epithelial cells of renal tubules and the gastrointestinal tract (36). GalCer is synthesized in the ER lumen and then trafficked into the Golgi where it can be modified further by sulfation or additional glycosylation before cell-surface presentation. Unlike GalCer-derived GSLs, structures based on GlcCer are ubiquitous. This GSL forms at the cytosolic face of the cis-Golgi and is translocated to the lumen of the Golgi via the Golgi stack trafficking process (37) to become a substrate for various glycosyltransferase enzymes and complexes. The addition of a Gal residue to GlcCer results in LacCer, which is the foundation for three additional classes of GSLs. First, the (neo-)lacto-series, or blood group series, begins with the addition of a β1-3-GlcNAc. Next, the Globo series is distinguished by the addition of an a1-4-Gal (also known as the Pk antigen). Third are the gangliosides, which are glycolipids that feature one or more sialic acid residues; membership in this group does not preclude inclusion in the lacto- or Globo series. Gangliosides are present in nearly all animal cells, but they are particularly prevalent in the plasma membranes of cells in the central nervous system (37).

Because of the importance of gangliosides in a variety of disease states and the volume of research devoted to them, the biosynthetic processes of this class of GSLs are described briefly. All gangliosides begin as LacCer except for GM4 (NeuAc-α2-3-GalCer). From LacCer, non-GM4 ganglioside biosynthesis continues down one of two branches: the asialo pathway (also called the o-pathway) through addition of a GalNAc residue, or into the “ganglioside proper” pathways (a-, b-, and c-pathways) through the addition of one or more sialic acid residues. GalNAc and Gal residues can be added as side chains that may be capped with additional sialic acids. Sialic acids appended to gangliosides can have subtle differences in structure that can result in drastic functional differences; a prime example is 9-O-acetylation of the terminal residue of GD3 that endows this potent inducer of apoptosis with anti-apoptotic properties (38). The N-terminal domains of the promiscuous glycosyltransferases responsible for the construction of the gangliosides specify the distribution of these enzymes within the Golgi stacks, which results in a differential expression pattern (37). A salvage pathway also exists for resynthesizing gangliosides, recycling them from their endosomal breakdown through the Golgi; this recycling pathway dominates in slowly dividing cells, whereas de novo synthesis dominates in highly mitotic cells.

GPI anchors

The discovery that phospholipase C could release alkaline phosphatase from lipid-linked structures on cellular surfaces (39) led to the identification of the glycophosphatidylinositol (GPI) membrane component (40). GPI structures are a synthetic tour de force of nature, combining lipids, carbohydrates, and proteins into a single macromolecule. Certain proteins require GPI anchoring to be functional; for example, Ly-6A/E-mediated T-cell activation is critically dependent on its GPI anchor (41), and folate uptake functions efficiently only when its receptors are GPI anchored (42).

The basic structure of the GPI-anchor (Fig. 6, maintained across all species studied thus far) begins with phosphatidylinositol (PI), which spans the external ER membrane leaflet linked to an inositol via a phosphodiester. An oligosaccharide chain, which is attached to the inositol, consists of GlcN (donated from a rapidly de-acetylated UDP-GlcNAc) and three linear Man residues (provided by Dol-P-Man donors). Finally, phos- phoethanolamine (P-EtN) is linked to the terminal Man residue (donated by phosphatidylethanolamine), resulting in the core EtN-P-Man3-GlcN-PI structure to which proteins are covalently linked [although not all GPI anchors ultimately bear a protein (43)]. In mammals, before the attachment of a protein, GPIs are completely assembled in the membrane of the ER by a series of enzymes that are PIG gene products (43). The topology of every biosynthetic step has not been elucidated. The synthetic process begins on the cytosolic face of the ER, and the attachment of protein occurs on the luminal face of the ER membrane (44), which suggests that a yet-to-be-discovered “flippase” participates in the production of GPI-anchored structures (45).

Phosphoethanolamine provides the attachment point for a protein via an amide bond between the C-terminal residue of the protein and the N-terminal of P-EtN (43). Proteins that are destined for GPI binding are targeted to the ER during their synthesis by an N-terminal signal and translocated to the ER lumen. They contain a C-terminal signal peptide, which upon removal exposes their acidic C-termini and allows attachment to GPI by ethanolamine through a transamidation reaction. GPI-anchored proteins belong to the type-1 class of the GPI structures, which have Man-a1-6-Man-α1-4-GlcN-α1-6-PI core linkages (other GPI structures have varying linkages between these core residues). After passing through the Golgi apparatus for further protein modification, the entire structure is translocated to the exterior leaflet of the plasma membrane.

Figure 6. Structure of a GPI anchor. Glycophosphatidylinositol (GPI) anchors are important functional structures on the cell surface. The fatty acid phosphatidylinositol is embedded in the exterior leaflet plasma membrane and features a tether consisting of a specific series of monosaccharides and phosphoethanolamine linked to the C-terminus of a protein.

Polysaccharides

Aside from the modification of proteins, lipids, and GPI anchors with branching oligosaccharide structures, mammals also assemble carbohydrates into much larger, linear polysaccharide structures. Despite losing the inherent complexity derived from branching, and being made from repeating units of only two monosaccharides, polysaccharides are nonetheless highly diverse through a series of postsynthetic modifications, primarily epimerization and sulfation reactions (46).

Polysaccharides generally exist outside of a cell, sometimes remaining attached to surface elements to form an interface between a cell and its surroundings, and sometimes secreted freely into the extracellular matrix (ECM). These sugars possess their own inherent functionality and are of critical importance to cellular function by modulating adhesion, migration, differentiation, and proliferation as well as by influencing angiogenesis and axonal growth. ECM polysaccharides become highly hydrated and thus serve as hydrogels for embedded fibrous ECM proteins, such as collagen, as well as scaffolds for signaling molecules such as growth factors. In mammals, four main classes of structural polysaccharides are all glycosamino-glycans (GAGs): hyaluronic acid (or hyaluronan), heparin (or heparan sulfate), keratin sulfate, and chondroitin/dermatan sulfate. Each group is now discussed briefly.

Figure 7. Metabolic oligosaccharide engineering of sialic acid. Exogenously supplied analogs of ManNAc or sialic acid can intercept the sialic acid biosynthetic pathway (at different points) and be displayed on the cell surface in place of the natural sialic acids Neu5Ac and Neu5Gc. The sampling of non-natural "R" groups shown here was selected from References 84, 92, 93 and 97-101 (note that not all "mix-and-match"' permutations of the indicated groups have been reported). Additional information on the enzymes shown can be found in the KEGG databases (see the Legend for Fig. 3).

Hyaluronan

Hyaluronan, or hyaluronic acid, is synthesized at the plasma membrane (rather than in the ER or Golgi apparatus) by one of three distinct hyaluronan synthases, which allows it to be easily secreted directly to the ECM (47). Hyaluronan is the simplest GAG, consisting of the repeating unit -GlcA-β1-3-GlcNAc-β1-4-; this GAG forgoes postsynthetic modification and remains unbound to surface proteins.

Heparin/heparan sulfate and chondroitin/dermatin sulfate

Heparin/heparan sulfate GAGs (HSGAGs) and condroitin/ dermatan sulfate GAGs (CSGAGs) share a common synthetic origin, with both being linked to a core protein through a specific

O-linked sequence (GlcA-β1-3-Gal-β1-3-Gal-P1-4-Xyl-P1-) at the same consensus sequence (-Ser-Gly/Ala-X-Gly-). The assembly of the tetrasaccharide linker begins in the ER where Xyl (from UDP-Xyl) is transferred to the hydroxyl group of the serine in the consensus sequence by a xylosyltransferase. The nascent glycoprotein then moves into the cis-Golgi where two Gal (by galactosyl transferase I and II) and a GlcA (by glucoronic acid transferase I) are attached sequentially to complete the tetramer.

Synthesis of the polysaccharide portion of these GAGs begins with the addition of GalNAc (or GlcNAc) and GlcA residues to the O-linked tetramer in an alternating fashion by multidomain glycosyltransferases (48). The addition of the first GalNAc or GlcNAc residue determines whether the GAG will belong to the heparan sulfate or condroitin sulfate family, respectively. HSGAGs consist of the repeating unit -GlcNAc-α1-4-GlcA-α/β1-4-, which is constructed by enzymes from the EXT gene family glycosyltransferases (49). CSGAGs have a different basic disaccharide repeat unit (-GalNAc-β1-4-GlcA-α/β1-3-), which contain GalNAc rather than GlcNAc and employ 1-3-rather than 1-4-glycosidic linkages between the repeating disaccharides; despite the differences in the monosaccharide building blocks used, CSGAG are also constructed from genes in the EXT family (48). When the HSGAG or CSGAG chain has grown to an appropriate length, it is acted upon by additional enzymes that impart structural uniqueness. 2-O-, 3-O-, and 6-O-sulfotransferases add sulfate groups at appropriate locations (50), N-deacetylase N-sulfotransferase can expose the amine groups of GalNAc, and C5 epimerase converts a portion of GlcA residues to IdoA. This epimerization results in the distinction between condroitin sulfate and dermatan sulfate (51).

Keratan Sulfate

Keratan sulfate differs from other GAGs in two major respects. First, it can be either N- or O-linked to the core protein (52). Second, its repeating disaccharide unit contains a Gal rather than one of the uronic acids in its disaccharide repeat. The basic repeating unit is -Gal-β1-4-GlcNAc-β1-3-, assembled by β1-4-galactosyl transferase (B4GALT1) and a β1-3-GlcNAc transferase (B3GNT1 or B3GNT2). Three classes of keratan sulfate are distinct in their protein linkage. KSI members are N-linked to an Asn of the protein; they are found primarily in the cornea and can be terminated with sialic acids, Gal, or GlcNAc. KSII members are O-linked to a Ser/Thr residue of the core protein; they are primarily found in cartilage, are highly sulfated, and are terminated by sialic acids. KSIII are found in brain tissue and have a unique linker between the keratan sulfate chain and the protein: a Man O-linked to a Ser of the protein.

Manipulating Mammalian Glycans—Early Steps Toward Sugar-Based Medicines

Although many aspects of glycans remain mysterious, mammalian glycosylation has now been elucidated well enough to provide a basic understanding of glycan biosynthesis, structure, and function that, combined with the realization that many disease states result from glycan abnormalities, have spurred efforts to develop sugar-based medicines. As modern medical research looks for ways to exploit the knowledge that has been gained regarding glycan biosynthesis to create new therapeutics, several challenges inherent in modifying glycosylation in living cells and tissues must be overcome (53). A particular obstacle to biologic intervention is the lack of template for carbohydrate structures akin to the DNA sequence that specifies primary amino acid sequences of proteins that motivates gene therapies. Similarly, the expense of de novo synthesis of complex oligosaccharides coupled with their notoriously poor pharmacologic properties make them nonideal drug candidates and hinders conventional synthetic approaches to drug development. Notwithstanding these limitations, both biology- and chemistry-based approaches remain enticing; moreover, merging these two disciplines into innovative “chemical biology” strategies seems particularly promising. This article concludes by discussing each of these topics briefly, after providing a short synopsis of the contributions of rapidly coalescing glycomics efforts to the study of mammalian glycosylation and development of human therapeutics.

Advances in technology and bioinformatics ease characterization

Efforts to manipulate glycans for medical purposes rely on knowing what oligosaccharide structures exist in health and disease, the biosynthetic machinery that builds these sugars, and specific molecular changes rendered by therapeutic intervention. In the past, the difficulty of characterizing glycans has made tackling even one of these tasks formidable. Now, however, bioinformatics has combined technological advances in carbohydrate analysis with the concept of global characterization found in genomics and proteomics to create the field of “glycomics”—the study of all the glycan structures produced by the cell. Modern methods using mass spectrometry, chromatography, nuclear magnetic resonance, and capillary electrophoresis have identified many carbohydrate structures (3, 54, 55). These techniques, along with high throughput arrays using lectins and other glycan-binding proteins (56), have resulted in a significant amount of information available in databases such as those available online from the German Cancer Research Center (http://www.glycosciences.de), the Consortium for Functional Glycomics (http://www.functionalglycomics.org), and the AFMB-CNRS of the University of Provence and the University of the Mediterranee (http://www.cazy.org). These resources complement automated methods for predicting function, structure, and localization of newly discovered glycans and glycan-related enzymes alongside their arrays of references, composition and spatial structures, and gathered NMR shift data (4). These developments are critical both for the glycobiology specialist undertaking further study of the intricacies of glycosylation and well as for the non-specialist, such as a physician, who seeks to apply glycobiology-based technologies in the clinic.

Biological approaches

Genetic approaches

Fascinating findings that viruses alter the glycome by regulating expression of host glycosyltransferases or by expressing their own glycosyltransferases (57) and forward genetics approaches that have identified glycan defects associated with specific genetic abnormalities (58) have motivated efforts to exploit modern genetic tools to manipulate glycosylation. Unfortunately, tools such as knockout mice to eliminate a specific biosynthetic enzyme often have manifold and severe effects such as early lethality (59). By contrast, in other situations, including the “aGal” knockout pig created to supply organs for xenotransplantation (60), the removal of a glycosyltransferase did not abolish production of the targeted oligosaccharide epitope. Notwithstanding these difficulties, the compelling links between defects in specific glycosylation enzymes and disease currently refractory to treatment, such as cancer (20), have ensured the continuation of research efforts to develop genetic methods to manipulate glycans.

Molecular biology approaches

In addition to upregulation or knock-down of biosynthetic elements, modern molecular biology offers techniques for more subtle manipulation of the glycosylation process. The biosynthesis of glycan structures relies on precisely localized enzymes for proper construction, for example, the localization of gly- cosylation enzymes within the ER and Golgi cisternae in the same sequence in which they act to modify oligosaccharide substrates (61). One way that this localization is achieved is based on the thickness of the membranes, which increases from the ER to the cis-, medial-, and trans-Golgi compartments; gly- cosyltransferase enzymes possess transmembrane domains of a length optimal to anchor them to a specific location in a cell’s secretory organelles (62). It is therefore possible to relocate an enzyme involved in glycosylation by swapping that enzyme’s native transmembrane region with a transmembrane domain of a different length and thereby changing substrate preference (63). Additionally, the stem region, located between the transmembrane and catalytic domains, can also be swapped to tune the activity of a glycosyltransferase (64). Although currently largely laboratory research tools, these studies point the way to a future where fine control over glycosylation may be possible by mix-and-matching the membrane, stem, and catalytic domains of glycan processing enzymes.

Synthetic approaches

Modern synthetic chemistry has been able to reproduce several glycan structures of considerable complexity and biomedical relevance. The pioneering example is the use of synthetic sialyl Lewis X for the treatment of reperfusion injury (65). For the past decade, much effort, which has been facilitated by automated synthesis (66), has focused on the creation of carbohydrate-based vaccines. It is possible to use synthetic carbohydrate analogs of viral and microbial surface polysaccharides as vaccines to elicit an immune response against the microorganism. In fact, because an “artificial” polysaccharide can be carefully designed through precise synthesis, this type of vaccine may be both safer and more effective at lower dosage [i.e., through multivalency (67, 68)] than a naturally derived vaccine such as that of a live or killed microbe that contains a mixture of glycoforms, some of which may be immunogenic (69). Synthetic polysaccharide vaccines have been recently developed for several targets, including Haemophilus influenza type b (70), human immunodeficiency virus (71), and various cancers (72, 73). In the future, as synthetic strategies are streamlined to become both technically and cost-effective, the possibilities of using carbohydrates to positively impact human health are numerous; for example, human breast milk contains a multitude of oligosaccharides that are distinct from other species such as the cow. Human-specific milk sugars are both developmentally important and have activity against pathogens (74), and the ability to supplement infant formula with these sugars would be valuable especially in the third world nations where malnutrition is endemic and infectious diseases are prevalent.

Chemical biology approaches

Combined chemical-biological synthesis

Combining tried-and-true synthetic methods with emerging chemoselective ligation methodology (75) and chemoenzymatic transformations that use the suite of enzymes cells employ for glycosylation (76) has led to the production of a multitude of carbohydrate structures. These hybrid approaches, exploiting biologic tools for programmable one-pot strategies (77), have several attractive features, including the ability to not only make a carbohydrate moiety but rather an entire glycoprotein (78) or glycolipid (79). Another important aspect of a synthetic strategy is that chemically distinct glycan structures can be produced allowing evaluation of the biological response of an individual glycoform, rather than an averaged response obtained when testing a mixture of the profusion of glycoforms found in nature. To illustrate, prion proteins from diseased and healthy cells have different glycan profiles that are proposed to be critical for disease progression (80), but this hypothesis is difficult to verify without the synthetic ability, which is afforded by the methods described herein, of producing testable quantities of individual prion glycoforms.

Small-molecule switches for controlling glycosylation

The emerging use of small molecules to control the activity of glycan processing enzymes (81) and direct biosynthetic traffic in the Golgi (82) constitutes an intriguing approach for modulating glycan synthesis. These efforts build on the modular nature of glycosyltransferases, where the membrane, stem, and catalytic domains can be swapped without loss of function. In particular, fusion proteins were created that combined the catalytic or localization domain of fucosyltransferase 1 (FUT1) with the rapamycin-binding proteins FKRP or FRB. Then, by exploiting rapamycin-mediated heterodimerization of these elements to control Golgi localization of both domains and thereby reconstitute FUT1 activity, Kohler and Bertozzi showed that cell-surface glycosylation could be altered (83). In the future, bringing a rich complement of biologic and chemical tools into play, as exemplified by this approach, will continue to drive progress toward glycan-based therapies.

Metabolic oligosaccharide engineering

“Metabolic oligosaccharide engineering” is an attractive alternative to the synthetic strategies outlined above not only because of its basic simplicity but also because it provides one of the few ways to alter glycosylation in living systems. This methodology, pioneered by the Reutter laboratory for sialic acid (84) (Fig. 7) and now extended to GalNAc (85, 86) and GlcNAc (87), is based on the remarkable ability of certain non-natural monosaccharide analogs to be metabolically incorporated into glycosylation pathways and to replace the corresponding sugar residue in the oligosaccharide complement of a cell.

The opportunities provided by metabolic oligosaccharide engineering can be divided into two essential classes. First, the intended target glycan can be endowed with a unique function. In particular, ManNAc analogs can be used to increase the repertoire of sialic acids, which is a family of over 50 natural sugars that nature uses to fine-tune function and structure (88), and thereby endow glycans with antiviral properties (89), enhance immunogenicity (90), modulate cell adhesion (91), or control stem cell fate (92). Alternatively, when the analog bears a chemical functional group unique to the cell surface, such as a ketone (93) or an azide (94), such sugars can act as “tags” for the delivery of genes (95), toxins (93), or imaging agents (96) by exploiting chemoselective ligation chemistry that has been developed to be compatible with physiologic conditions (75).

Acknowledgments

The authors thank S.-G. Sampathkumar for critical comments on the manuscript and the National Institutes of Health (1R01CA112314-01A1), the National Science Foundation (QSB-0425668), and the Arnold and Mabel Beckman Foundation for financial support.

References

  1. Gabius H-J, Siebert H-C, Andre S, Jimenez-Barbero J, Rudiger H. Chemical biology of the sugar code. ChemBioChem. 2004; 5:740-764.
  2. Ratner DM, Adams EW, Disney MD, Seeberger PH. Tools for glycomics: Mapping interactions of carbohydrates in biological systems. ChemBioChem. 2004; 5:1375-1383.
  3. Campbell CT, Yarema KJ. Large-scale approaches for glycobiology. Gen. Biol. 2005; 6:236.
  4. von der Lieth CW, Lutteke T, Frank M. The role of informatics in glycobiology research with special emphasis on automatic interpretation of MS spectra. Biochim. Biophys. Acta 2006; 1760:568-577.
  5. Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta, 1999; 1473:4-8.
  6. Bell GI, Burant CF, Takeda J, Gould GW. Structure and function of mammalian facilitative sugar transporters. J. Biol. Chem. 1993; 268:19161-19164.
  7. Joost H-G, Bell GI, Best JD, Birnbaum MJ, Charron MJ, Chen YT, et al. Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators. Am. J. Physiol. Endocrinol. Metab. 2002; 282:E974-E976.
  8. Stuart CA, Yin D, Howell ME, Dykes RJ, Laffan JJ, Ferrando AA. Hexose transporter mRNAs for GLUT4, GLUT5, and GLUT12 predominate in human muscle. Am. J. Physiol. Endocrinol. Metab. 2006; 291:1067-1073.
  9. Wu X, Freeze HH. GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms. Genomics 2002; 80:553-557.
  10. Chen H, Wang Z, Sun Z, Kim EJ, Yarema KJ. Mammalian glycosylation: an overview of carbohydrate biosynthesis In: Handbook of Carbohydrate Engineering. Yarema KJ, ed. 2005. Francis & Taylor/CRC Press, Boca Raton, FL. p. 1-48.
  11. Varki A. Loss of N-glycolylneuraminic acid in humans: mechanisms, consequences, and implications for hominid evolution. Am. J. Phys. Anthropol. 2002; 116:54-69.
  12. Zachara NE, Hart GW. Cell signaling, the essential role of O-GlcNAc! Biochim. Biophys. Acta 2006; 1761:599-617.
  13. Berninsone PM, Hirschberg CB. Nucleotide sugar transporters of the Golgi apparatus. Curr. Opin. Struct. Biol. 2000; 10:542-547.
  14. Pels Rijcken WR, Hooghwinkel GJ, Ferwerda W. Pyrimidine metabolism and sugar nucleotide synthesis in rat liver. Biochem. J. 1990; 266:777-783.
  15. Capasso JM, Hirschberg CB. Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/ nucleoside monophosphate antiports in the Golgi apparatus membrane. Proc. Natl. Acad. Sci. U.S.A. 1984; 81:7051-7055.
  16. Hirschberg CB. Golgi nucleotide sugar transport and leukocyte adhesion deficiency II. J. Clin. Invest. 2001; 108:3-6.
  17. Kamiyama S, Suda T, Ueda R, Suzuki M, Okubo R, Kikuchi N, et al. Molecular cloning and identification of 3’-phosphoadeno- sine 5’-phosphoadenosine’-phosphosulfate transporter. J. Biol. Chem. 2003; 278:25958-25963.
  18. Helenius A, Aebi M. Intracellular functions of N-linked glycans. Science 2001; 291:2364-2369.
  19. Freeze HH, Aebi M. Altered glycan structures: the molecular basis of congenital disorders of glycosylation. Curr. Opin. Struct. Biol. 2005; 15:490-498.
  20. Dennis JW, Granovsky M, Warren CE. Glycoprotein glycosylation and cancer progression. Biochim. Biophys. Acta, 1999; 1473:21-34.
  21. Helenius J, Aebi M. Transmembrane movement of dolichol linked carbohydrates during N-glycoprotein biosynthesis in the endoplasmic reticulum. Semin. Cell. Dev. Biol. 2002; 13:171- 178.
  22. Spiro RG. Glucose residues as key determinants in the biosynthesis and quality control of glycoproteins with N-linked oligosaccharides. J. Biol. Chem. 2000; 275:35657-35660.
  23. Bause E. Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem. J. 1983; 209:331-336.
  24. Burda P, Aebi M. The dolichol pathway of N-linked glycosylation. Biochim. Biophys. Acta 1999; 1426:239-257.
  25. Parodi AJ. Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem. J. 2000; 348:1-13.
  26. Kumar R, Yang J, Larsen RD, Stanley P. Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation. Proc. Natl. Acad. Sci. U.S.A. 1990; 87:9948-9952.
  27. Tan J, D’Agostaro GAF, Bendiak B, Reck F, Sarkar M, Squire JA, et al. The human UDP-N-acetylglucosamine: α-6-D-manno- side-β-1,2-N-acetylglucosaminyltransferase II gene (MGAT2): cloning of genomic DNA, localization to chromosome 14q21, expression in insect cells and purification of the recombinant protein. Eur. J. Biochem. 1995; 231:317-328.
  28. Brockhausen I, Narasimhan S, Schachter H. The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI. Biochimie 1988; 70:1521-1533.
  29. Hang HC, Bertozzi CR. The chemistry and biology of mucin-type O-linked glycosylation. Bioorg. Med. Chem. 2005; 13:5021- 5034.
  30. Torres CR, Hart GW. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J. Biol. Chem. 1984; 259:3308-3317.
  31. Zachara NE, Hart GW. The emerging significance of O-GlcNAc in cellular regulation. Chem. Rev. 2002; 102:431-438.
  32. Love DC, Hanover JA. The hexosamine signaling pathway: deciphering the “O-GlcNAc code”. Science STKE 2005; 312(re13).
  33. Hakomori S-I. The glycosynapse. Proc. Natl. Acad. Sci. U.S.A. 2002; 99:225-232.
  34. Michel C, van Echten-Deckert G. Conversion of dihydroceramide to ceramide occurs at the cytosolic face of the endoplasmic reticulum. FEBS Lett. 1997; 416:153-155.
  35. van Echten G, Sandhoff K. Ganglioside metabolism. Enzymology, topology, and regulation. J. Biol. Chem. 1993; 268:5341- 5344.
  36. Degroote S, Wolthoorn J, van Meer G. The cell biology of glycosphingolipids. Semin. Cell. Dev. Biol. 2004; 15:375-387.
  37. Maccioni HJF, Giraudo CG, Daniotti JL. Understanding the stepwise synthesis of glycolipids. Neurochem. Res. 2002; 27:629-636.
  38. Malisan F, Franchi L, Tomassini B, Ventura N, Condo I, Rippo MR, et al. Acetylation suppresses the proapoptotic activity of GD3 ganglioside. J. Exp. Med. 2002; 196:1535-1541.
  39. Low MG, Finean JB. Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C. Biochem. J. 1977; 167:281-284.
  40. Futerman AH, Low MG, Ackermann KE, Sherman WR, Silman I. Identification of covalently bound inositol in the hydrophobic membrane-anchoring domain of Torpedo acetylcholinesterase. Biochem. Biophys. Res. Commun. 1985; 129:312-317.
  41. Su B, Waneck GL, Flavell RA, Bothwell ALM. The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T 63. cell activation. J. Cell. Biol. 1991; 112:377-384.
  42. Ritter TE, Fajardo O, Matsue H, Anderson RG, Lacey SW. Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake. Proc. Natl. Acad. Sci. U.S.A. 1995; 92:3824-3828.
  43. McConville MJ, Menon AK. Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids. Mol. Membr. Biol. 2000; 17:1-16.
  44. Vidugiriene J, Menon AK. The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum. J. Cell. Biol. 1994; 127:333-341.
  45. Chatterjee S, Mayor S. The GPI-anchor and protein sorting. Cell Mol. Life Sci. 2001; 58:1969-1987.
  46. Esko JD, Lindahl U. Molecular diversity of heparan sulfate. J. Clin. Invest. 2001; 108:169-173.
  47. Itano N, Kimata K. Mammalian hyaluronan synthases. IUBMB Life 2002; 54:195-199.
  48. Sasisekharan R, Venkataraman G. Heparin and heparan sulfate: biosynthesis, structure and function. Curr. Opin. Chem. Biol. 2000; 4:626-631.
  49. Kim B-T, Kitagawa H, Tamura J-i, Saito T, Kusche-Gullberg M, Lindahl U, et al. Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α-1,4-N-acetylglucos- aminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2001; 98:7176-7681.
  50. Sugahara K, Kitagawa H. Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans. Curr. Opin. Struct. Biol. 2000; 10:518-527.
  51. Silbert JE, Sugumaran G. Biosynthesis of chondrotin/dermatan sulfate. IUBMB Life 2002; 54:177-186.
  52. Funderbergh JL. Keratan sulfate biosynthesis. IUBMB Life 2002; 54:187-194.
  53. Dove A. The bittersweet promise of glycobiology. Nat. Biotechnol. 2001; 19:913-917.
  54. von der Lieth CW, Bohne-Lang A, Lohmann KK, Frank M. Bioinformatics for glycomics: status, methods, requirements and perspectives. Brief Bioinform. 2004; 5:164-178.
  55. Morelle W, Michalski J-C. Glycomics and mass spectrometry. Curr. Pharm. Des. 2005; 11:2615-2645.
  56. Pilobello KT, Krishnamoorthy L, Slawek D, Mahal LK. Development of a lectin microarray for the rapid analysis of protein glycopatterns. ChemBioChem. 2005; 6:985-989.
  57. Markine-Goriaynoff N, Gillet L, Van Etten JL, Korres H, Verma N, Vanderplasschen A. Glycosyltransferases encoded by viruses. J. Gen. Virol. 2004; 85:2741-2754.
  58. Stanley P, Raju TS, Bhaumik M. CHO cells provide access to novel N-glycans and developmentally regulated glycosyltransferases. Glycobiology 1996; 6:696-699.
  59. Furukawa K, Sato T. β-1,4-Galactosylation of N-glycans is a complex process. Biochim. Biophys. Acta 1999; 1473:54-66.
  60. Milland J, Christiansen D, Sandrin MS. α1,3-Galactosyltransferase knockout pigs are available for xenotransplantation: Are glycosyltransferases still relevant? Immunol. Cell Biol. 2005; 83:687-693.
  61. Colley KJ. Golgi localization of glycosyltransferases: more questions than answers. Glycobiology 1997; 7:1-13.
  62. Sasai K, Ikeda Y, Tsuda T, Ihara H, Korekane H, Shiota K, et al. The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction. J. Biol. Chem. 2001; 276:759-765.
  63. de Graffenried CL, Bertozzi CR. Golgi localization of carbohydrate sulfotransferases is a determinant of L-selectin ligand biosynthesis. J. Biol. Chem. 2003; 278:40282-40295.
  64. de Graffenried CL, Bertozzi CR. The stem region of the sulfotransferase GlcNAc6ST-1 is a determinant of substrate specificity. J. Biol. Chem. 2004; 279:40035-40043.
  65. Schermerhorn ML, Nelson DP, Blume ED, Phillips L, Mayer 84. JE Jr. Sialyl LewisX oligosaccharide preserves myocardial and endothelial function during cardioplegic ischemia. Ann. Thorac. Surg. 2000; 70:890-894.
  66. Seeberger PH, Werz DB. Automated synthesis of oligosaccharides as a basis for drug discovery. Nat. Rev. Drug Discov. 2005; 4:751-763.
  67. Kiessling LL, Pohl S. Strength in numbers: non-natural polyvalent carbohydrate derivatives. Chem. Biol. 1996; 3:71-77.
  68. Ragupathi G, Koide F, Livingston PO, Cho YS, Endo A, Wan Q, et al. Preparation and evaluation of unimolecular pentavalent and hexavalent antigenic constructs targeting prostate and breast cancer: a synthetic route to anticancer vaccine candidates. J. Am. Chem. Soc. 2006; 128:2715-2725.
  69. Werz DB and Seeberger PH. Carbohydrates as the next frontier in pharmaceutical research. Chem Eur J, 2005; 11(11):3194-3206.
  70. Verez-Bencomo V, Fernandez-Santana V, Hardy E, Toledo ME, Rodriguez MC, Heynngnezz L, et al. A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. Science 2004; 305:522-525.
  71. Pashov AD, Plaxco J, Kaveri SV, Monzavi-Karbassi B, Harn D, Kieber-Emmons T. Multiple antigenic mimotopes of HIV carbohydrate antigens - relating structure and antigenicity. J. Biol. Chem. 2006; 281:29675-29683.
  72. Deshpande PP, Danishefsky SJ. Total synthesis of the potential anticancer vaccine KH-1 adenocarcinoma antigen. Nature 1997; 387:164-166.
  73. Keding SJ, Danishefsky SJ. Prospects for total synthesis: a vision for a totally synthetic vaccine targeting epithelial tumors. Proc. Natl. Acad. Sci. U.S.A. 2004; 101:11937-11942.
  74. Hueso P, Martin-Sosa S, Martin M-J. Role of milk carbohydrates in preventing bacterial adhesion. In: Handbook of Carbohydrate Engineering. Yarema KJ, ed. Taylor & Francis (CRC Press), Boca Raton, FL. p. 141-175.
  75. Lemieux GA, Bertozzi CR. Chemoselective ligation reactions with proteins, oligosaccharides and cells. Trends Biotechnol. 1998; 16:506-513.
  76. Meldal M, St Hilaire PM. Synthetic methods of glycopeptide assembly, and biological analysis of glycopeptide products. Curr. Opin. Chem. Biol. 1997; 1:552-563.
  77. Koeller KM, Wong CH. Complex carbohydrate synthesis tools for glycobiologists: enzyme-based approach and programmable one-pot strategies. Glycobiology 2000; 10:1157-1169.
  78. Pratt MR, Bertozzi CR. Synthetic glycopeptides and glycoproteins as tools for biology. Chem. Soc. Rev. 2005; 34:58-68.
  79. Pan Y, Chefalo P, Nagy N, Harding C, Guo Z. Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines. J. Med. Chem. 2005; 48:875-883.
  80. Rudd PM, Endo T, Colominas C, Groth D, Wheeler SF, Harvey DJ, et al. Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc. Natl. Acad. Sci. U.S.A. 1999; 96:13044-13049.
  81. de Graffenried CL, Laughlin ST, Kohler JJ, Bertozzi CR. A small-molecule switch for Golgi sulfotransferases. Proc. Natl. Acad. Sci. U.S.A. 2004; 101(16715-20): 16715-16720.
  82. Kohler JJ, Czlapinski JL, Laughlin ST, Schelle MW, de Graffenried CL, Bertozzi CR. Directing flux in glycan biosynthetic pathways with a small molecule switch. ChemBioChem. 101. 2004; 5:1455-1458.
  83. Kohler JJ, Bertozzi CR. Regulating cell surface glycosylation by small molecule control of enzyme localization. Chem. Biol. 2003; 10:1303-1311.
  84. Kayser H, Zeitler R, Kannicht C, Grunow D, Nuck R, Reutter W. Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors. J. Biol. Chem. 1992; 267:16934-16938.
  85. Hang HC, Bertozzi CR. Ketone isoteres of 2-N-acetamido- sugars as substrates for metabolic cell surface engineering. J. Am. Chem. Soc. 2001; 123:1242-1243.
  86. Dube DH, Prescher JA, Quang CN, Bertozzi CR. Probing mucin-type O-linked glycosylation in living animals. Proc. Natl. Acad. Sci. U.S.A. 2006; 103:4819-4824.
  87. Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain. Proc. Natl. Acad. Sci. U.S.A. 2004; 2004(36):13132-13137.
  88. Angata T, Varki A. Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective. Chem. Rev. 2002; 102:439-469.
  89. Keppler OT, Horstkorte R, Pawlita M, Schmidt C, Reutter W. Biochemical engineering of the N-acyl side chain of sialic acid: biological implications. Glycobiology 2001; 11:11R-18R.
  90. Liu T, Guo Z, Yang Q, Sad S, Jennings HJ. Biochemical engineering of surface a2,8 polysialic acid for immunotargeting tumor cells. J. Biol. Chem. 2000; 275:32832-32836.
  91. Villaviecencio-Lorini P, Laabs S, Danker K, Reutter W, Horstkorte R. Biochemical engineering of the acyl side chain of sialic acids stimulates integrin-dependent adhesion of HL60 cells to fibronectin. J. Mol. Med. 2002; 80:671-677.
  92. Sampathkumar S-G, Li AV, Jones MB, Sun Z, Yarema KJ. Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat. Chem. Biol. 2006; 2:149-152.
  93. Mahal LK, Yarema KJ, Bertozzi CR. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 1997; 276:1125-1128.
  94. Saxon E, Bertozzi CR. Cell surface engineering by a modified Staudinger reaction. Science 2000; 287:2007-2010.
  95. Lee JH, Baker TJ, Mahal LK, Zabner J, Bertozzi CR, Wiemar DF, et al. Engineering novel cell surface receptors for virus-mediated gene transfer. J. Biol. Chem. 1999; 274:21878-21884.
  96. Lemieux GA, Yarema KJ, Jacobs CL, Bertozzi CR. Exploiting differences in sialoside expression for selective targeting of MRI contrast reagents. J. Am. Chem. Soc. 1999; 121:4278-4279.
  97. Gross HJ. Fluorescent CMP-sialic acids as a tool to study the specificity of the CMP-sialic acid carrier and the glycoconjugate sialylation in permeabilized cells. Eur. J. Biochem. 1992; 203:269-275.
  98. Jacobs CL, Goon S, Yarema KJ, Hinderlich S, Hang HC, Chai DH, et al. Substrate specificity of the sialic acid biosynthetic pathway. Biochemistry 2001; 40:12864-12874.
  99. Oetke C, Brossmer R, Mantey LR, Hinderlich S, Isecke R, Reutter W, et al. Versatile biosynthetic engineering of sialic acid in living cells using synthetic sialic acid analogues. J. Biol. Chem. 2002; 277:6688-6695.
  100. Kim EJ, Sampathkumar S-G, Jones MB, Rhee JK, Baskaran G, Yarema KJ. Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acetylmannosamine (ManNAc) analogs in Jurkat (human T-lymphoma-derived) cells. J. Biol. Chem. 2004; 279:18342-18352.
  101. Han S, Collins BE, Bengtson P, Paulson JC. Homo-multimeric complexes of CD22 revealed by in situ photoaffinity protein- glycan crosslinking. Nat. Chem. Biol. 2005; 1:93-97.

Further Reading

Chatterjee S, Mayor S. The GPI-anchor and protein sorting. Cell. Mol. Life. Sci. 2001; 58:1969-1987.

Eisenhaber B, Maurer-Stroh S, Novatchkova M, Schneider G, Eisenhaber F. Enzymes and auxiliary factors for GPI lipid anchor biosynthesis and post-translational transfer to proteins. BioEssays 2003; 25:367-385.

Grogan MJ, Pratt MR, Marcaurelle LA, Bertozzi CR. Homogeneous glycopeptides and glycoproteins for biological investigation. Annu. Rev. Biochem. 2001; 71:593-634.

Helenius A, Aebi M. Roles of A-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 2004; 73:1019-1049.

Hinderlich S, Oetke C, Pawlita M. Biochemical engineering of sialic acids.In: Handbook of Carbohydrate Engineering. Yarema KJ, ed. Francis & Taylor/CRC Press, Boca Raton, FL. p. 387-405.

Ikezawa H. Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol. Pharm. Bull. 2002; 25:409-417.

Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 1985; 54:631-664.

Spiro RG. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 2002; 12:43R-56R.

Van den Steen P, Rudd PM, Dwek RA, Opdenakker G. Concepts and principles of O-linked glycosylation. Crit. Rev. Biochem. Mol. Biol. 1998; 33:151-208.

See Also

Glycans in Information Storage and Transfer

Glycans, Mammalian

Glycosylation of Proteins in the Golgi Compartment

Glycomics, Major Techniques

Glycan Therapeutics, Engineering of