neural cell adhesion molecule N-acetyl neuraminic acid nerve growth factor

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3 From Page xi Lennarz W.J. and Hart G.W., eds Guide to techniques in glycobiology. Methods Enzymol., vol Academic Press, San Diego, California. Iozzo R., ed Proteoglycans: Structure, biology and molecular interactions. Marcel Dekker, Inc., New York. From Contributors, Page xiii Jeffrey Esko, Professor of Cellular and Molecular Medicine Associate Director, Glycobiology Research and Training Center University of California, San Diego, La Jolla, California Adriana Manzi, Director, Analytical Research and Development/Quality Control Nextran Inc., an affiliate of Baxter Healthcare Corporation, San Diego, California From Abbreviations, Page xvii N-CAM Neu5Ac NGF neural cell adhesion molecule N-acetyl neuraminic acid nerve growth factor From Chapter 1, Page 6 FIGURE 1.4. Recommended symbols and conventions for drawing glycan structures. The example

4 From Chapter 1, Page 7 FIGURE 1.5. Basic core structures of the common classes of animal glycans. (For the monosaccharide symbol code used for the depictions, see Figure 1.4.) From Chapter 1, Page 9 polylactosamine and is therefore attached to a N- or O-glycan core rather than a typical proteoglycan core region. One type of glycosaminoglycan, hyaluronan (GlcNAcβ1-4GlcA) n, appears to exist primarily as a free sugar chain, unattached to any aglycone. The glycosaminoans From Chapter 1, Page 10 FIGURE 1.6. Biosynthesis, utilization, and turnover of a common monosaccharide. This schematic shows the biosynthesis, fate, and turnover of one common monosaccharide constituent of animal glycans, galactose. Although small amounts of galactose can be taken up from the outside of the cell, most is either synthesized de novo from glucose or recycled from degradation of glycoconjugates in the lysosome. The schematic presents a simplified view of the generation of the UDP sugar nucleotide, its equilibrium state with UDP-glucose, and its uptake and utilization in the Golgi apparatus for synthesis of new glycans. (Solid lines) Biochemical pathways; (dashed lines) pathways for the trafficking of membranes and glycans. From Chapter 4, Page 42 runs for most of the length of one surface of the protein. This cleft is astonishingly large, considering that lysozyme has only 129 amino acids, and is capable of accommodating a hexasaccharide and cleaving it into a disaccharide product and a tetrasaccharide product.

5 From Chapter 6, Page 77 N-acetylglucosamine (22,23) Synthesis of UDP-GlcNAc begins with the formation of GlcN-6-P from Fru-6-P by transamidation using glutamine as the NH 2 donor. Glucosamine-6-P is then N-acetylat From Chapter 7, Page 93 but appears optimal on N-glycans bearing the GlcNAcT-V branch. GlcNAcT-V requires the prior activity of GlcNAcT-II. GlcNAcT-III and GlcNAcT-V are sometimes exclusive, as the action of GlcNAcT-III inhibits the action of GlcNAcT-V. GlcNAcT-VI action is uncommon, but it may require prior branching by GlcNAcT-II and GlcNAcT-V. From Chapter 7, Page 93 N-glycosylation sites on the same protein may contain different glycan structures, a finding referred to as microheterogeneity. It appears that factors other than protein sequence may influence N-glycan diversification. Such factors may include sugar nucleotide metabolism, transport rates in the ER and Golgi, and the localization of glycosyltransferases in the From Chapter 8, Page 102 FIGURE 8.1. Human small intestinal mucin. Large numbers of O-glycan chains (wavy lines) are found throughout the polypeptide (encoded MUC2 gene), also termed the apomucin. A few N- glycosylation sites are indicated (tridents). Disulfide bridges may occur in an intra- or intermolecular fashion. (Reprinted with permission, from [56] Toribara et al ) From Chapter 8, Page 103 FIGURE 8.2. Multiple polypeptide GalNAcTs initiate O-glycosylation in all multicellular organisms studied. At least eight highly homologous genes exist in the genomes of Caenorhabditis elegans, Mus musculus, and Homo sapiens. (Adapted, with permission, from [55] Marth 1996 [ Oxford University Press].) O-GLYCAN DIVERSIFICATION IN VERTEBRATES: CORE SUBTYPE FORMATION (29 35) From Chapter 8, Page 104 The glycosyltransferase responsible is known as the Core 1 β1-3 galactosyltransferase (Core 1 GalT) (Figure 8.3). None of the cdnas encoding the Core 1 GalTs have been identified, although it appears based on enzyme activities that multiple Core 1 GalT enzymes may exist in vertebrates. Like the polypeptide GalNAcT family, the Core 1 GalTs appear to

6 From Chapter 8, Page 105 Core 2-type O-glycans can be generated by addition of GlcNAc to the GalNAc in a β1-6 linkage. The production of Core 2 O-glycans requires the Core 1 structure as a substrate, so the Core 2 structure also contains the Core 1 structure. For this reason, use of the term Core 1 O-glycans refers to an O-glycan in which Core 2 GlcNAcT has not acted. Recent From Chapter 8, Page 106 FIGURE 8.4. Core 3 and Core 4 O-glycan subtype formation is controlled by the activity of the Core 3 GlcNAcT and Core 4 GlcNAcT enzymes. Further O-glycan biosynthesis can yield biantennary forms sometimes similar to those of the Core 2 subtypes. Additional linkages may subsequently occur (vertical arrow). From Chapter 8, Page 107 FIGURE 8.6. Early and alternate pathways in O-glycan biosynthesis leading to the formation of tumor-associated (T) antigens. Specific sialyltransferases can act to produce the sialyl Tn and disialyl T antigens. These appear to be biosynthetic dead ends (boxed) that cannot be further modified. High levels of various T antigens are frequently associated with cancer cells (see Chapter 35).

7 From Chapter 8, Pages O-glycans perform a role in the formation of the ABO blood group antigens, a system that has clinical relevance in blood transfusions, although glycolipids are also known to carry these antigens (see Chapter 16). The blood group antigens are enriched on the erythrocyte surface and their diversity is a product of mutations in a glycosyltransferase encoded by the H locus. Mutations in another glycosyltransferase generate either null alleles (O) or actual changes in substrate specificity (A and B) (see Chapter 16). Antibodies to specific blood group oligosaccharides have previously defined blood types by agglutination activities, but the endogenous roles of these O-glycan structures are not presently known. O-GLYCAN FUNCTIONS IN ENDOGENOUS LECTIN-LIGAND INTERACTIONS (44 57) O-glycans have been reported to function in sperm binding to the egg. The mammalian egg coat (the zona pellucida) contains a large number of O-glycans, as well as some N-glycans. Removal of egg N- glycans by glycosidase treatment does not destroy sperm binding, but loss of O-glycans following mild alkali treatment ablates sperm binding. Of those O-glycans found on the mammalian egg, the ZP3 glycoprotein of the mouse zona pellucida has been reported to have the primary role of the sperm receptor. Analyses of the O-glycan structures on ZP3 have revealed terminal α1-3gal residues on O-glycans. Although this structure was previously believed to be responsible for sperm-egg bind From Chapter 9, Page 119 TABLE 9.1. Names and abbreviations for major core structures of vertebrate glycosphingolipids Name (Series) Abbreviation Core Structure Lacto (LcOSe 4 ) Galβ3GlcNAcβ3Galβ4Glcβ1Ceramide Lactoneo (LcnOSe 4 ) Galβ4GlcNAcβ3Galβ4Glcβ1Ceramide Globo (GbOSe 4 ) GalNAcβ3Galα4Galβ4Glcβ1Ceramide Isoglobo (GbiOSe 4 ) GalNAcβ3Galα3Galβ4Glcβ1Ceramide Ganglio (GgOSe 4 ) Galβ3GalNAcβ4Galβ4Glcβ1Ceramide Muco (MucOSe 4 ) Galβ3Galβ3Galβ4Glcβ1Ceramide Gala (GalOSe 2 ) Galα4Galβ1Ceramide Sulfatide 3-O-Sulfo-Galβ1Ceramide From Chapter 9, Page 120 FIGURE 9.4 Pathways for the biosynthesis of the ganglio series glycosphingolipids. Note that the action of the GalNAc transferase commits the molecules to further extension along one of four different pathways; the consequences of genetic disruption of this enzyme are discussed in the text. Further additions of sialic acids occur in some cell types and species, giving polysialogangliosides (not shown).

8 From Chapter 9, Page 121 ner that would be indistinguishable from that of molecules that were endogenously synthesized. The extent to which such intercellular transfer can take place in vivo is not known. When fluorescently labeled ceramide is added to culture medium, it is taken up into the From Chapter 9, Page 121 It has also been suggested that gangliosides are involved in thermal adaptation of neuronal membranes. The data suggest a general rule that the lower the environmental temperature the more polar is the composition of brain gangliosides. Studies with model bilayer membranes also indicate that gangliosides can modulate basic membrane properties in a thermosensitive manner. Hakomori and colleagues have also provided evidence that glycosphingolipids can mediate low-affinity but highspecificity carbohydrate-carbohydrate From Chapter 11, Page 153 the polymer has formed (cf. Chapter 6). The GalNAc 6-sulfotransferase, GalNAc 4-sulfotransferase, and iduronic acid 2-O sulfotransferase have been purified and cloned. Progress in this area is expected to accelerate as more genome sequences become available from various organisms. CS assembly can occur on virtually all proteoglycans, depending on the cell in which the core protein is expressed. The major proteoglycans that typically contain CS or DS chains From Chapter 15, Page 200 FIGURE Biosynthesis and turnover of the N-acyl group of sialic acids. The general pathways for biosynthesis, activation, and transfer of N-acetylneuraminic acid are shown. The step at which the N-acetyl group can be hydroxylated is indicated. Page 215 FIGURE Modification of exposed GlcNAc moieties by galactosylation. Modification by β1-4-linked galactose residues (top) is generally found in all mammalian tissues. This reaction is cata

9 Page 216 FIGURE Examples of polylactosamine chains on various glycans. Page 219 FIGURE Type-4 A, B, and O(H) blood group structures. LEWIS BLOOD GROUP STRUCTURES (72 94) Page 223 The Lewis blood group antigens correspond to a structurally similar set of α1-3(4)-fucosylated glycan structures (Figure 16.14). The term Lewis refers to the family name of indi Page 224 the Le (a b ) phenotype. In contrast, in individuals who are Lewis-negative nonsecretors, type-1 precursors are not substituted by either the Lewis or the Secretor fucosyltransferases, accounting for absence of Le a,le b, or H structures, and their Le (a b ) phenotype. Expression of Le a and Le b molecules, and the Lewis α1-3/1-4 fucosyltransferase, is

10 Page 224 FIGURE Representative type-1 and type-2 Lewis structures. (Top) Type-2-based structures; (bottom) type-1-based structures. Type-1 and type-2 structures differ in the linkage of the outermost Gal (β1-3 and β1-4, respectively) and in the linkage of the fucose moiety to the internal GlcNAc (α1-4 and α1-3, respectively). R represents N-linked, O-linked, or glycolipid-based substructures. Page 225 FIGURE Lewis blood group phenotypes. The structures of the Lewis blood-group-active glycolipids are determined by the presence or absence of the Se locus α1-2 fucosyltransferase and the Lewis locus α1-4 fucosyltransferase. R represents underlying glycoconjugates. Page 225 Le x (SSEA-1) and Le y molecules and forms of the Le a and Le x determinants that are sialylated and or sulfated (see Figure 16.14). These structures are formed through the actions of one or more α1-3 fucosyltransferases in addition to the Lewis α1-3(4) fucosyltransferase.

11 Page 227 FIGURE Antigens of the P blood group system. Page 227 the P precursor (P k ) is not present. However, p phenotype red cells express low levels of a P-like antigen reactivity distinct from that of P antigen. This reactivity likely corresponds to the recognition of a different complex glycolipid whose structure is currently unknown. These considerations assume that the P, P 1, and P k transferases represent the products of three Page 228 FIGURE P 1 antigen biosynthesis via paragloboside. The P blood group antigens have also been assigned a role as a receptor for the human parvovirus B19. This virus causes erythema infectiosum and leads to congenital anemia and hydrops fetalis following infection in utero. It is also associated with transient aplastic crisis in patients with hemolytic anemia and with cases of pure red cell aplasia and chronic anemia in immunocompromised individuals. Parvovirus B19 replication is restricted to erythroid progenitor cells; an adhesive interaction between the virion and P-antigen-active glycolipids is involved in viral infection of erythroid progenitors. Individuals with the p blood group phenotype are apparently resistant to parvovirus B19 infection. Page 230 FIGURE Forssman antigen biosynthesis. Globoside serves as the substrate for the Forssman α1-3 N-acetylgalactosaminyltransferase (α1-3 GalNAcT) that forms globopentosylceramide, also termed the Forssman glycolipid. THE FORSSMAN ANTIGEN ( ) Page 230 The Forssman antigen, also known as globopentosylceramide, is a glycolipid structure formed by the addition of GalNAc in α1-3 linkage to the terminal GalNAc residue of globoside (Figure 16.20). There

12 Page 233 FIGURE Production of the Sd a or CT antigen and the glycolipid G M2. Page 236 Structures bearing α2-3 sialic acid linkages have also been implicated in contributing to bacterial pathogenesis. For example, α2-3 sialic acid structures support adhesion of H. pylori, the spirochete implicated in the pathogenesis of gastritis, gastric ulcers, and lymphoma of the gastrointestinal tract mucosa. However, it remains to be determined if these in vitro observations have a physiological correlate. In contrast, there is strong evidence for a physiological role for the ganglioside G M1 (G M1a ; Galβ1-3GalNAcβ1-4[Siaα2-3]Galβ1-4Glcβ1-Cer) as a receptor for cholera toxin, produced by Vibrio cholerae, and heat-labile enterotoxin (LT-1), produced by enterotoxigenic E. coli (see Chapter 28). Cholera toxin is responsible for the severe enteropathogenicity that accompanies infection with V. cholerae. Page 237 α2-6-sialylated STRUCTURES (139,140,149, ) Sialic acid in α2-6 linkage is expressed by various vertebrate cells and tissue types and displayed on a wide variety of glycoproteins and glycolipids. Synthesis of α2-6-linked sialic moieties is directed by members of a family of at least five different α2-6 sialyltransferases (see Figure 16.24). The genes of these have been defined by molecular cloning FIGURE Synthesis of α2-6-sialylated termini on N-glycans, O-glycans, and glycolipids (and see Chapters 8 and 9) by the ST6GalNAc series of sialyltransferases. Enzymes in parentheses contribute at relatively low levels in vitro to the reactions indicated.

13 Page 237 approaches (ST6Gal-I, ST6GalNAc-I, ST6GalNAc-II, ST6GalNAc-III, and ST6GalNAc- IV). A gene corresponding to a sixth activity, ST6GlcNAc-I, has not yet been genetically isolated. Surveys of tissues and cells in a variety of mammals, and some lower vertebrates, indicate that α2-6-sialylated glycans may be found in various (but not all) cell types, as they appear less ubiquitous than the α2-3-linked sialic-acid-bearing glycans. hepatocytes and lymphocytes and is responsible for α2-6 sialylation of serum glycoproteins and glycoproteins of the antigen receptor complex. In contrast, the α2-6-sialylated products of ST6GalNAc-I and ST6GalNAc-II and the enzymes themselves are restricted to structures displayed on O-glycans. The ST6GalNAc-III enzyme appears to use glycosphingolipid precursors as the preferred acceptor, whereas the ST6GalNAc-IV enzyme is responsible for substitution of core N-acetylgalactosamine moiety on O-glycans. Few definitive functions have been assigned to α2-6-linked sialic acid modifications. As noted above (and see Chapter 28), α2-6-linked sialic acid serves as a receptor for human Page 240 Closed Diamond Deleted FIGURE Structure and synthesis of polysialic acid on glycolipids. Enzymes in parentheses contribute at low levels in vitro to the reactions indicated. Page 241 FIGURE Sulfation of α2-3-sialylated, α1-3 fucosylated glycans represented by the sialyl Le x tetrasaccharide have been observed at the 6-hydroxyl positions of the galactose residue and the GlcNAc residue. Both may contribute to L-selectin ligand activity. The biosynthesis of these structures, the enzymes that participate in this process, and their functional attributes are discussed further in Chapter 26.

14 From Chapter 17, Page 254 FIGURE A generic glycosylation reaction. A glycosyltransferase utilizes a glycosyl donor and an acceptor substrate. Glycosyl donors can include nucleotide sugars and dolichol-phos From Chapter 17, Page 255 GENERAL PROPERTIES OF GLYCOSYLTRANSFERASE REACTIONS (1,2,4,5,7,12) Most glycosyltransferase-dependent transglycosylations involve a divalent cation as a cofactor (typically Mg ++ or Mn ++ ), and the enzymes tend to be most active in the ph range of 5.0 to 7.0, which reflects ph values found in various parts of the ER-Golgi-plasmalemma pathway. Glycosyltransferases typically exhibit Michaelis-Menten constants (K m s) for nucleotide sugar substrates in the low micromolar range, when assayed in vitro. These val From Chapter 17, Page 256 FIGURE Strict acceptor substrate requirement of the human B blood group α1-3 galacto

15 From Chapter 17, Page Lindahl U., Kusche-Gullberg M., and Kjellén L Regulated diversity of heparan sulfate. J. Biol. Chem. 273: Beyer T.A., Rearick J.I., Paulson J.C., Prieels J.P., Sadler J.E., and Hill R.L Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences. J. Biol. Chem. 254: Watkins W.M Biochemistry and genetics of the ABO, Lewis, and P blood group systems. Adv. From Chapter 19, Page 287 FIGURE Lipid-linked oligosaccharide biosynthesis and mutants in yeast. Lipid-linked oligosaccharide synthesis and transfer to protein in the ER is genetically defined by a wealth of functional mutations that effect lipid-linked oligosaccharide structure and transfer to protein. Some of the steps in the synthesis of the earliest precursors such as Man-6-P and Man-1-P have also been detected but are not shown here. Defects in dolichol biosynthesis and recycling are expected to also produce mutations that would reduce glycosylation efficiency. (Dark gray) Mannose residues that originate from GDP-Man; (light gray) mannose residues from dolichylphosphomannose. (Modified, with permission, from [3] Burda and Aebi 1999 [ Elsevier Science].) From Chapter 19, Page Kuwabara P.E Worming your way through the genome. Trends Genet. 13: Chen S., Zhou S., Sarkar A., Spence A., and Schachter H Expression of three Caenorhabditis elegans N- acetylglucosaminyltransferase I genes during development. J. Biol. Chem. 274: Haslam S.M., Coles G.C., Munn E.A., Smith T.S., Smith H.F., Morris H.R., and Dell A Haemonchus contortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylat

16 From Chapter 20, Page 314 FIGURE Structures of a high-mannose N-glycan and three complex N-glycans found in plant glycoproteins. From Chapter 24, Page 364 TABLE Established and putative members of the I-type lectin family Minimal Alternate Tissue/Cell type Domain carbohydrate Lectin name distribution structure structure(s) recognized Siglecs Siglec 1 sialoadhesin macrophages in spleen, (V) 1 -(C2) 16 Siaα2-3Galβ1-3(4)GlcNAclymph nodes, and Siaα2-3Galβ1-3GalNAcbone marrow Siglec 2 CD22 B cells (V) 1 -(C2) 6 Siaα2-6Galβ1-4GlcNAc- Siglec 3 CD33 myeloid cell lineage (V) 1 -(C2) 1 Siaα2-3Galβ1-3(4)GlcNAc- Siaα2-3Galβ1-3GalNAc- Siglec 4a MAG PNS (V) 1 -(C2) 4 Siaα2-3Galβ1-3GalNAc Siglec 4b SMP Schwann cells in quail (V) 1 -(C2) 4 Siaα2-3Gal- Siglec 5 granulocytes and (V) 1 -(C2) 3 Siaα2-3Gal- and Siaα2-6-Galmonocytes Non-Siglecs PECAM platelets heparin P0 PNS N-CAM PNS and CNS (V) 1 (C2) 5 SO 3 GlUAβ1-3Galβ1-R (HNK1 epitope) a high-mannose oligosaccharide a endothelium, etc. ICAM-1 blood cells, (C2) 5 hyaluronan (GlcNAcβ1-3GlUAβ1-4) n leukosialin (sialylated mucin) a CD48 activated B cells (V) 1 -(C2) 1 heparin and heparan sulfate a In these cases, the evidence for specific binding involving carbohydrates is indirect.

17 From Chapter 24, Page 364 lectins, whereas the subset recognizing sialylated structures is termed the Siglecs (sialic acid/immunoglobulin/lectin) and has been numbered sequentially by common agreement. This chapter reviews structural and functional data about this group of lectins, concentrating on the Siglecs, about which the most is known. COMMON FEATURES OF SIGLECS Domain Structure and Genetics(13-21) The primary amino acid sequence of the I-type lectins indicates that they are members of the IgSF, containing an amino-terminal V-set domain followed by a variable number of C2-set domains (V l - C2 n ) (see Table 24.1). The C2-set domains are a variation of the more common C1-set domains and are believed to represent a primordial motif predating the From Chapter 24, Page 365 FIGURE Sequence alignments of the first two domains of the murine forms of CD22, sialoadhesin, MAG, and CD33 are shown, with regions of identity included in boxes and regions of similarity are shaded. Above the sequences are the regions of predicted and confirmed (for sialoadhesin) β strands. Asterisks indicate the residues known to be involved in binding of sialyllactose in sialoadhesin and are identified in Figure The leader peptides have been deleted from each sequence. From Chapter 29, Page 446 In contrast to HS, one might expect that a better consensus would exist for HA-binding proteins due to the uniformity of the repeats in HA ([GlcAβ1-3GlcNAcβ1-4] n ). Indeed, a consensus sequence for HA binding was deduced by comparing a number of HA-binding proteins: BX 7 B, where B is argi From Chapter 29, Page 447 Heparin is solely the product of mast cells, and although it has proven to be of great therapeutic use, the natural oligosaccharide that participates in the control of antithrombin/thrombin has not been identified. Endogenous heparan sulfate present on endothelial cells can contain small amounts of antithrombin binding sequence, unlike heparan sulfates from most other tissues and cells that contain none. The location of these binding From Chapter 29, Page 448 sequences, however, appears to be on the ablumenal side of the cells, which would not be exposed to blood except after injury. The nature of the endogenous activator of antithrombin remains unknown.

18 From Chapter 29, Page 449 The structure of FGF-2 cocrystallized with a heparin hexasaccharide has been obtained (Figure 29.5). The heparin fragment ([GlcNS6Sα1-4IdoA2Sα1-4] 3 ) was helical and bound to a region of the bfgf surface containing residues Asn-28, Arg-121, Lys-126, and Gln-135 and an additional binding site formed by Lys-27, Asn-102, and Lys-136. The linear seg From Chapter 32, Page 482 The PMM2 locus encodes an enzyme that catalyzes an essential step in the biosynthesis of GDP- Man and Dol-P-Man (Figure 32.2). These two compounds are essential substrates for the mannosyltransferases required for the synthesis of the lipid-linked precursor for N-glycosylation (Figure 32.2). In CDGS type Ia, a deficiency of phosphomannomutase activity reduces the levels of GDP-Man and Dol-P-Man. The reduced levels of these compounds reduce synthesis of Glc 3 Man 9 GlcNAc 2 -PP-Dol, the physiological substrate for oligosaccharyltransferase. Reductions in GDP-Man and Dol-P-Man From Chapter 32, Page 483 It is important to point out that Dol-P-Man is also required for the synthesis of the four mannose residues of glycophosphotidylinositol anchors (see Chapter 10) and is the major source of GDP-Fuc required for terminal fucosylation (see Leukocyte Adhesion Deficiency From Chapter 32, Page 486 FIGURE 32.3 N-glycans that accumulate in CDGS type II and in HEMPAS. (Normal) N-glycans that are present on red cell bands 3 and 4.5; n may be from 1 to several, and represents the number of lactosamine units within the polylactosamine chain. CDGSII glycans are not modified From Chapter 32, Page 490 Structural analyses of the band-3 and band-4.5 glycans indicate that these vary to some significant extent among different individuals with HEMPAS. In some such patients, the abnormal glycans feature a truncation of the antenna attached to the α1-6-linked mannose residue of the trimannosyl core structure in the N-glycan (Figure 32.3). This structure is the immediate synthetic precursor to GlcNAcT-II, and its excessive accumulation has implied a defect in the expression or activity of this enzyme in some HEMPAS patients (Figure 32.3). Although it has been reported that the cells in some HEMPAS patients exhibit 70 90% reduction in GlcNAcT-II activity, the pathophysiological relevance of

19 From Chapter 32, Page 496 From Chapter 32, Page Marquardt T., Brune T., Lühn K., Zimmer K.-P., Kömer C., Fabritz L., van der Werft N., Vormoor J., Freeze H.H., Louwen F., Biermann B., Harms E., von Figura K., Vestwever D., and Koch H.G Leukocyte adhesion deficiency II syndrome, a generalized defect in fucose metabolism. J. Pediatr. 13: Marquardt T., Lühn K., Srikrishna G., Freeze H.H., Harms E., and Vestweber D Correction of leukocyte adhesion deficiency type II (LAD II) with oral fucose. Blood 94: Crookston J.H., Crookston M.C., Burnie K.L., Francombe W.H., Dacie J.V., Davis J.A., and Lewis S.J Fukuda M.N., Bothner B., Scartezzini P., and Dell A Isolation and characterization of poly-n-acetyllactosaminylceramides accumulated in the erythrocytes of congenital dyserythropoietic anemia type II patients. Chem. Phys. Lipids 42: From Chapter 33, Page 507 New Art FIGURE Site-directed recombination and conditional gene mutagenesis in vivo. Mice bear From Chapter 33, Page 510 New Art FIGURE Branch specificity is observed in N- and O-glycan structure-function relationships.

20 From Chapter 38, Page 590 Once the number and relative ratios of glycan forms present in a glycoprotein are known, it is possible to calculate from the analytical chromatography result whether a sufficient quantity of individual glycans can be prepared to completely identify their structures by physicochemical approaches (i.e., NMR and MS). Quantity of purified material permitting, the best choice is to perform 1 H NMR analysis first. Because this method is nondestructive, the same sample can later be used for other, destructive approaches (e.g., MS and methylation analysis). The limitations on the use of NMR spectroscopy are the cost of equipment and the level of expertise required for interpreting NMR spectra. However, access to high-field (i.e., 500 MHz and above) NMR spectrometers fitted with very sensitive probes (e.g., Nano-NMR probes) allows one to obtain 1 H NMR profiles of individual HPLC fractions using minute quantities of sample (2 5 nmoles per oligosaccharide). As an example, the 1 H NMR spectrum of a mixture of two trisialyl triantennary oligosaccharides obtained from bovine fetuin is shown in Figure From Chapter 38, Page 593 FIGURE Methylation analysis: A chemical approach to elucidating the glycosidic linkage positions in glycans.

21 From Chapter 38, Page 596 FIGURE MALDI-TOF mass spectra of N-glycan mixtures obtained from 5 µg of bovine fetuin after treatment at ph 7.5 with (A) PNGase F and neuraminidase; (B) PNGase F, neuraminidase, and β1-4 galactosidase; (C) PNGase F, neuraminidase, β1-4 galactosidase, and β- N-acetylglucosaminidase (adapted, with permission, from Mechref and Novotny, Anal. Chem. 70: [1998].) The m/z values measured for the various peaks are compatible with the monosodium adducts of oligosaccharides with the given schematic structures (for explanation of symbolic notation, see Chapter 1). From Chapter 41, Page 632 TABLE Examples of enzyme-based inhibitors as potential therapeutics Clinical situation Drug/Mechanism Company Influenza A infection GS4104 (neuraminidase inhibitor) Gilead/Hoffman LaRoche Influenza A infection Zanamivir (Relenza) (neuraminidase inhibitor) Glaxo Wellcome/Biota Tumors and hepatitis swainsonine (α-mannosidase II inhibitor) GlycoDesign Glycolipid accumulation in lysosomal storage diseases glycosyltransferase inhibitors Oxford GlycoSciences Diabetes Acarbose (intestinal α-glucosidase inhibitor) Bayer Flea infestation in pets Lufenuron (Program ) (chitin synthesis inhibitor) Ciba-Geigy