alzheimer sialic

The Chemical Neurobiology of Carbohydrates

Heather E. Murrey and Linda C. Hsieh-Wilson*

Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology,Pasadena, California 91125

Received February 4, 2008

Contents

1. Introduction 17082. Sialic Acids 17092.1. Structure 17092.2. Neurobiological Functions 17102.2.1. R(2-3)-Sialic Acid and Myelin-Associated

Glycoprotein1710

2.2.2. Polysialic Acid 17102.3. Chemical Neurobiology of Sialic Acid 17112.3.1. Synthetic Sialic Acid Derivatives: Probing

the Specificity of MAG Interactions1711

2.3.2. Development of MAG Antagonists withTherapeutic Potential

1711

2.3.3. Synthetic Mimics of R(2-8)-Linked PSAfor Nerve Regeneration

1712

2.3.4. Metabolic Labeling To RemodelCell-Surface Sialic Acid Interactions

1712

3. R-L-Fucose 17143.1. Structure and Biosynthesis 17143.2. Neurobiological Functions 17143.2.1. Neuronal Development 17143.2.2. Learning and Memory 1715

3.3. Chemical Approaches for Studying L-Fucose 17153.3.1. Deoxygalactose Analogues 17153.3.2. Glycopolymers and Imaging Probes 17163.3.3. Metabolic Labeling Using Alkynyl or Azido

Fucose Analogues1716

3.3.4. Summary of Fucosyl Oligosaccharides 17164. O-GlcNAc Glycosylation 17164.1. Structure and Biological Functions 17164.2. Neurobiological Functions of O-GlcNAc 17174.3. Chemical Tools To Study O-GlcNAc

Glycosylation1717

4.3.1. Chemoenzymatic Labeling of O-GlcNAcProteins

1717

4.3.2. Metabolic Labeling of O-GlcNAc Proteins 17194.3.3. Methods for Mapping Exact Glycosylation

Sites1719

4.3.4. Monitoring O-GlcNAc Dynamics 17205. Glycosaminoglycans 17225.1. Structure and Diversity 17225.2. Neurobiological Functions 17235.2.1. Neuronal Development 17235.2.2. Axon Guidance 17235.2.3. Spinal Cord Regeneration 1723

5.3. Challenges to the Study of GAGs 17235.4. Synthetic Molecules for Probing

Structure-Activity Relationships1723

5.4.1. Synthesis of Glycosaminoglycans 17235.4.2. Effects of HS and DS Molecules on

Neuronal Growth1724

5.4.3. Neuroactive Small-Molecule ChondroitinSulfates

1724

5.5. Carbohydrate Microarrays for StudyingGAG-Protein Interactions

1724

5.5.1. Oligosaccharide Microarrays 17245.5.2. Polysaccharide Microarrays 1725

5.6. Glycosaminoglycan-Based Therapeutics 17255.6.1. Prion Diseases 17255.6.2. Alzheimers Disease 17255.6.3. Future Challenges 1726

6. Summary and Future Directions 17267. Acknowledgments 17268. References 1726

1. IntroductionThe cell surface displays a complex array of oligosaccha-

rides, glycoproteins, and glycolipids. This diverse mixtureof glycans contains a wealth of information, modulating awide range of processes such as cell migration, proliferation,transcriptional regulation, and differentiation.15 Glycosyla-tion is one of the most ubiquitous forms of post-translationalmodification, with more than 50% of the human proteomeestimated to be glycosylated.6 Glycosylation adds anotherdimension to the complexity of cellular signaling andexpands the ability of a cell to modulate protein function.The structural complexity of glycan modifications rangesfrom the addition of a single monosaccharide unit topolysaccharides containing hundreds of sugars in branchedor linear arrays.7 This chemical diversity enables glycans toimpart a vast array of functions, from structural stability andproteolytic protection to protein recognition and modulationof cell signaling networks.8,912

Emerging evidence suggests a pivotal role for glycans inregulating nervous system development and function. Forinstance, glycosylation influences various neuronal processes,such as neurite outgrowth and morphology, and maycontribute to the molecular events that underlie learning andmemory.7,13,14 Glycosylation is an efficient modulator of cellsignaling and has been implicated in memory consolidationpathways.1518 Genetic ablation of glycosylation enzymesoften leads to developmental defects and can influencevarious organismal behaviors such as stress and cognition.1924Thus, the complexity of glycan functions help to orchestrate

* To whom correspondence should be addressed. E-mail: [email protected].

Chem. Rev. 2008, 108, 170817311708

10.1021/cr078215f CCC: $71.00 2008 American Chemical SocietyPublished on Web 05/02/2008

proper neuronal development during embryogenesis, as wellas influence behaviors in the adult organism.

The importance of glycosylation is further highlighted bydefects in glycan structures that often lead to human disease,as exhibited by congenital disorders of glycosylation(CDG).2529 These are usually inherited disorders resultingfrom defects in glycan biosynthesis, which are accompaniedby severe developmental abnormalities, mental retardation,and difficulties with motor coordination. Such disordershighlight the importance of glycan biosynthesis in humanhealth and development. Because therapeutic treatments arecurrently limited, investigations into the structure-activityrelationships of glycans, as well as disease-associatedalterations to glycan structure, are crucial for developingstrategies to combat these diseases.

Understanding the structure-function relationships ofglycans has been hampered by a lack of tools and methodsto facilitate their analysis. In contrast to nucleic acids and

proteins, oligosaccharides often have branched structures, andtheir biosynthesis is not template-encoded. As such, thecomposition and sequence of oligosaccharides cannot beeasily predicted, and genetic manipulations are considerablyless straightforward. Analytical techniques for investigatingoligosaccharide composition, sequence, and tertiary structureare still undergoing development and are far from routine,unlike methods for DNA and protein analysis. Lastly, glycanstructures are not under direct genetic control and, thus, areoftenheterogeneous.Thisheterogeneitycomplicatesstructure-function analyses by traditional biochemical approaches thatrely on the isolation and purification of glycans from naturalsources.

The problems associated with oligosaccharide analysishave hindered efforts to understand the biology of oligosac-charides yet have given chemists a unique opportunity todevelop new methods to overcome these challenges. Thedevelopment of chemical tools for the analysis of glycanstructure and function is essential to advance our understand-ing of the roles of glycoconjugates in regulating diversebiological processes. In this review, we will highlight theemerging area of glyconeurobiology with an emphasis oncurrent chemical approaches for elucidating the biologicalfunctions of glycans in the nervous system.

2. Sialic Acids

2.1. StructureSialic acids participate in a multitude of biologically

interesting phenomena, including cell-cell recognition,adhesion, and intracellular signaling events.3032 Originallyknown as neuraminic acid (Neu) and its derivatives, sialicacids are a family of R-keto acids containing a nine-carbonbackbone.32 The most well-known members of the sialic acidfamily include N-acetylneuraminic acid (Neu5Ac), N-gly-colylneuraminic acid (Neu5Gc), and deaminoneuraminic acid(KDN) (Figure 1). In addition to these basic forms, morethan 50 distinct sialic acid structures have been identified innature, arising from acetylation, methylation, lactylation,sulfation, and phosphorylation of the C-4, C-5, C-7, C-8, orC-9 hydroxyl groups.

Sialic acids exist predominantly as terminal monosaccha-rides linked to galactose residues in glycan chains throughR(2-3)- or R(2-6)-linkages. They can also form a ho-mopolymer of R(2-8)-linked sialic acid in mammals, termedpolysialic acid (PSA).33,34 As discussed below, each glyco-form dictates a unique function to the glycoproteins andglycolipids expressing these sugars. Sialic acids have histori-cally received much attention due to their participation incell-cell recognition events and the pathogenesis of diseasessuch as cancer,3537 inflammatory disease,3840 and viralinfection.4144 The development of sialic acid analogues asinhibitors or probes for biomedical research has led to

Heather E. Murrey received a B.A./M.S. degree in biochemistry fromBrandeis University in 2000. She then conducted research at theWhitehead Institute for Biomedical Research in the laboratories of PeterS. Kim and Harvey F. Lodish. She is currently pursuing a Ph.D. at Caltechunder the direction of Linda C. Hsieh-Wilson. Her graduate studies havefocused on the role of fucosyl oligosaccharides in neuronal communicationand development.

Linda C. Hsieh-Wilson received her B.S. degree in chemistry from YaleUniversity in 1990. She then earned her Ph.D. degree in 1996 from theUniversity of California at Berkeley, where she worked with Peter G.Schultz. After completing postdoctoral studies in neurobiology at TheRockefeller University with Nobel Laureate Paul Greengard in 2000, shejoined the faculty at the California Institute of Technology, where she iscurrently Associate Professor of Chemistry. In 2005, she was appointeda Howard Hughes Medical Institute Investigator. Her research group worksat the interface of organic chemistry and neurobiology to study the rolesof carbohydrates and their associated proteins in transcription, neuronalsignaling, and brain development.

Figure 1. Common structures of sialic acid derivatives: neuraminicacid (Neu), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminicacid (Neu5Gc), and deaminoneuraminic acid (KDN).

Chemical Neurobiology of Carbohydrates Chemical Reviews, 2008, Vol. 108, No. 5 1709

significant advances in our understanding of this importantfamily of carbohydrates. Here, we will discuss some of theroles of sialic acids in neurobiology and chemical approachesthat have provided insight into their functions.

2.2. Neurobiological Functions2.2.1. R(2-3)-Sialic Acid and Myelin-AssociatedGlycoprotein

Sialic acid is often expressed as R(2-3)-linked sialic acidin the nervous system, a carbohydrate motif recognized bythe Siglec (sialic acid-binding immunoglobulin-like lectin)family of proteins. Human Siglecs include at least 13members, each containing a common V-set immunoglobulindomain that interacts with sialic acid.45 One interaction thathas been extensively studied is the binding of myelin-associated glycoprotein (MAG; also known as Siglec-4) withR(2-3)-sialic acid. MAG is a 100-kDa integral membraneglycoprotein that is expressed myelinating glia cells.46,47 Itis involved in regulating the formation and maintenance ofmyelin48 and has been suggested to inhibit nerve regenerationin the adult central nervous system (CNS).4951 Micedeficient in MAG display delayed myelination,52 defects inthe organization of periaxonal space,53 and subtle morpho-logical abnormalities of myelin sheaths.52 The interactionsof MAG with sialic acid-containing glycosphingolipids,known as gangliosides, have been extensively studied andhave contributed to our understanding the role of MAG inmyelin formation and neural regeneration.

MAG preferentially binds the glycan structure Neu5AcR-(2-3)Gal(1-3)GalNAc,54 which is expressed on cell-surface gangliosides and O-glycans of glycoproteins.47Gangliosides represent the major source of sialic acidexpression in the brain. MAG binds with high affinity andspecificity to the major brain gangliosides GD1a and GT1b,as well as the polysialoganglioside GQ1bR, a minor gan-glioside expressed on cholinergic neurons (Figure 2). Diges-tion of gangliosides purified from bovine brain with neuramini-dase, an enzyme that cleaves sialic acid residues, eliminatedthe binding of MAG to these gangliosides, demonstratingthe importance of the sialic acid moiety in mediatingMAG-ganglioside interactions.5557

Studies suggest that the association of MAG with sialicacid-containing gangliosides plays an important functionalrole in neuronal growth. The ability of MAG to inhibit neuriteoutgrowth in Vitro is blocked by treatment of cerebellargranule neurons with neuraminidase or with the glucosyl-

ceramide synthase inhibitor P4, which prevents synthesis ofall glycosphingolipids.55 Moreover, mice lacking the glyco-syltransferase gene GalNAcT (UDP-N-acetylgalactosamine:GM3/GD3 N-acetylgalactosaminyltransferase) do not expresscomplex gangliosides such as GD1a and GT1b and, as aconsequence, exhibit axon degeneration and gross dysmyeli-nation.58,59 These mice also display progressive behavioralabnormalities consistent with neurodegenerative disease, suchas defects in balance, reflexes, and motor coordination.59Thus, detailed knowledge of MAG and its interactions withsialylated glycans may enhance our understanding of my-elinating disorders such as multiple sclerosis and provideopportunities to enhance axon regeneration after CNS injuryor disease.

2.2.2. Polysialic Acid

In the brain, PSA is expressed primarily on the proteinneural cell adhesion molecule (NCAM).6062 NCAM playscritical roles in both nervous system development andmemory formation, regulating processes such as cell adhe-sion, axon targeting and fasciculation, neuronal migration,synaptic plasticity, and synaptogenesis.60,61,6370 PSA-NCAMis highly expressed in the embryonic brain7173 and is foundin the adult brain in areas that retain a high degree ofplasticity and neurogenesis, such as the hippocampus,olfactory bulb, and hypothalamus.7477

Although the molecular mechanisms underlying PSAfunction are not well understood, PSA is thought to modulatecell-cell adhesion by attenuating homophilic NCAM-NCAMinteractions. The large steric bulk and hydration shell of thecarbohydrate chain increase the intercellular space by 10-15m, reducing trans NCAM-NCAM interactions acrossapposing cells.78 In addition, PSA modulates the interactionsof NCAM with other proteins, such as heparan sulfateproteoglycans involved in the formation and remodeling ofhippocampal synapses.79 The PSA chains on NCAM havealso been proposed to play a role in some neuropsychiatricdisorders. For example, expression of PSA-NCAM issignificantly reduced in the hippocampus of schizophrenicpatients and may contribute to the complex symptomsassociated with the disease.8082 Moreover, PSA has beenimplicated in the etiology of Alzheimers disease, asPSA-NCAM-positive granule cells are increased in thehippocampus of Alzheimers patients and are associated withdisorganization of PSA-positive fibers.83 Finally, PSA mayalso regulate neuronal function through NCAM-independent

Figure 2. Structures of gangliosides that bind to MAG. Neu5Ac ) N-acetylneuramic acid; Gal ) galactose; GalNAc ) N-acetylgalactosamine;Glc ) glucose; Cer ) ceramide.

1710 Chemical Reviews, 2008, Vol. 108, No. 5 Murrey and Hsieh-Wilson

mechanisms. For example, PSA has been suggested to actas a competitive antagonist of the NMDA receptor, anionotropic glutamate channel involved in synaptic transmis-sion,84 thereby preventing glutamate-induced excitotoxicity.85

Despite intriguing roles for sialic acid-containing glycans,the molecular mechanisms underlying their diverse functionsin the brain remain largely unknown. As described below,chemical approaches to access and manipulate sialic acidstructures have expanded our understanding of the neuro-biological roles of sialic acid and promise to continue toadvance the field.

2.3. Chemical Neurobiology of Sialic Acid2.3.1. Synthetic Sialic Acid Derivatives: Probing theSpecificity of MAG Interactions

Synthetic sialic acid analogues have been used to elucidatethe molecular determinants important for MAG-gangliosideinteractions. The C-9 hydroxyl group represents a keyrecognition element: substitution of this group with hydrogen,halogen, or thiol groups attenuated the association of MAGwith Neu5Ac (Figure 3, compounds 1-5). Interestingly, anamino group at C-9 enhanced binding to MAG by 3-fold,suggesting the importance of a hydrogen donor at thisposition (compound 6).86 The C-5 N-acetyl group of Neu5Acwas also found to be critical for MAG binding, although itis not always required for interaction with other Siglecs.Replacement of this group with an N-propanoyl, N-ami-noacetyl, or N-thioacetyl moiety enhanced binding of sialicacid to MAG by up to 4-fold (compounds 7-9). Thecorresponding halogenated derivatives were all found toincrease the binding to MAG (compounds 10-13), with themonofluorinated derivative achieving a 17-fold increase inpotency. In contrast, amino substitution at the C-5 positionsignificantly attenuated binding to MAG.86 Together, thesestudies highlight key interactions between MAG and the C-9hydroxyl and C-5 N-acetyl groups of sialic acid.

In addition to probing monosaccharide variants, numerousoligosaccharide derivatives have been synthesized and tested

for binding to MAG. These structures mimic naturallyoccurring ganglioside structures such as GD1a (Figure 2).Consistent with previous studies, substitution of the C-9hydroxyl of Neu5Ac with a methyl group within thetrisaccharide Neu5AcR(2-3)Gal(1-4)Glc attenuated bind-ing to MAG by 5-fold, again highlighting the importance ofthe glycerol side chain.87 These results are consistent withan X-ray crystal structure of the Siglec sialoadhesin com-plexed with sialyllactose, in which the C-9 hydroxyl groupof NeuAc forms a hydrogen bond with the amide backboneof Leu-107.88 Although these proteins are distinct, it isconceivable that their mode of binding to sialic acid wouldbe conserved across Siglec family members. In contrast toC-9, the C-7 and C-4 hydroxyls do not appear to contributesubstantially to the binding energy of MAGsialic acidinteractions.87 The C-7 deoxy derivative of Neu5AcR(2-3)-Gal((1-4)Glc-2-azidoethyl exhibited only slightly en-hanced binding to MAG (1.5-fold), whereas the C-4 deoxyderivative showed slightly decreased binding (2-fold). How-ever, both the C-7 and C-4 hydroxyls appeared to be criticalfor binding when placed in the context of a polyvalentarray.57 Thus, valency and cell-surface presentation mayreflect another facet of the complex regulation and specificityof Siglec-ganglioside interactions.

Synthetic oligosaccharide derivatives have also providedinsight into the importance of specific glycosidic linkagesand other residues within the structure. MAG was found tobind 5-fold better to R(2-3)-linked Neu5Ac than to R(2-6)-linked Neu5Ac in synthetic trisaccharides.87 Interestingly,replacement of Neu5Ac in a pentasaccharide structure withthe naturally occurring sialic acid KDN led to a 6.5-foldincrease in MAG binding,87 suggesting that other sialic acidforms may bind MAG in ViVo. In addition to contacts withterminal sialic acid residues, internal sugars were also foundto be important for MAG interactions. For instance, substitu-tion of the C-4 hydroxyl group of galactose in Neu5AcR(2-3)-Gal(1-4)Glc with a hydrogen atom enhanced binding toMAG by 2.3-fold. Changing this residue to GalNAc, addingan O-methyl substituent at C-6, or exchanging the ringoxygen to an N-methyl or N-butyl functionality decreasedthe potency of the trisaccharide.87 Modifications of the thirdglucose residue to N-acetylglucosamine (GlcNAc) alsodecreased the binding properties of the molecules. Varioussubstitutions of the N-acetyl group, such as N-phthaloyl orN-octanoyl substituents, increased the potency of the com-pounds, which reflects the potential for a hydrophobicinteraction with MAG at this site.87 Lastly, pentasaccharidesof the structure Neu5AcR(2-3)Gal(1-4)AllNAc(1-3)-Gal(1-4)Glc-2-(trimethylsilyl)ethyl (AllNAc ) N-acetyl-allosamine) were found to increase binding above thetrisaccharide Neu5AcR(2-3)Gal(1-4)Glc by 6-fold, sug-gesting even more extensive contacts between MAG and theinterior residues of large glycan structures.87

Together, studies using synthetic analogues have illustratedhow subtle perturbations to the sialic acid core structure canhave significant effects on protein binding. As describedbelow, such studies may facilitate the design of novelsynthetic inhibitors of MAG function with therapeuticpotential.

2.3.2. Development of MAG Antagonists with TherapeuticPotential

The importance of MAG-ganglioside interactions fornerve regeneration and myelination has inspired the design

Figure 3. Synthetic sialic acid analogues tested for binding toMAG. Positions important for MAG interactions are shown in red.


and synthesis of small molecules capable of disrupting thoseinteractions. Such molecules have the potential to enhancenerve regeneration by blocking the inhibitory effects of MAGon neurite outgrowth. Below, we provide some examples ofsmall molecule antagonists that exhibit activity in cellularregeneration models.

Paulson and co-workers examined the interactions ofmonovalent sialic acid derivatives with MAG and otherSiglec family members.89 Over 25 derivatives representingmost of the major sialic acid structures found on glycopro-teins and glycolipids were tested. The most potent inhibitorof MAG-ganglioside interactions was the disialyl structureNeu5AcR(2-3)Gal(1-3)[Neu5AcR(2-6)]GalNAcR-O-ThrOCH3 (Figure 4A), which exhibited an IC50 value of 0.3M. This compound showed greater than 12000-fold en-hanced potency relative to Neu5Ac for inhibiting MAG-sialic acid interactions.89

The disialyl structure above and other potent inhibitorssuch as Neu5AcR(2-3)Gal(1-3)GalNAc were subse-quently tested for their ability to attenuate MAG-mediatedinhibition of neurite outgrowth.90 When rat cerebellar granuleneurons (CGN) are cultured on a substratum of myelin-extracted proteins, they project fasciculated axons and clustertogether, leaving the majority of the substrata bare. This formof neuronal growth inhibition is mediated primarily by MAG.The sialosides relieved the MAG-dependent inhibition ofCGN neurons, enhancing nerve regeneration in a dose-dependent manner and proportional to their relative bindingaffinities for MAG.90 The most potent compound, the disialylstructure, completely reversed the inhibition induced byMAG. Thus, synthetic glycans can effectively enhance neuriteoutgrowth in Vitro and, when used in combination with othertreatments, may provide a means to improve functional recoveryafter neuronal injury. The ability to compare various Siglecfamily members against a large number of sialoside structureshas also revealed the specificity of Siglecs for differentcarbohydrate epitopes and may help to fine-tune the develop-ment of selective MAG antagonists.

Many oligosaccharide-based inhibitors are syntheticallychallenging to produce and can suffer from poor pharma-cokinetics. As an alternative to this approach, Ernst and co-workers generated structurally simplified mimics of theganglioside GQ1bR. In particular, the Gal and GalNAcresidues in the trisaccharide Neu5AcR(2-3)Gal(1-3)GalNAcwere replaced with an R-linked benzyl ether moiety, andaromatic residues were positioned on the glycerol side chain(Figure 4B). Despite its smaller size, this compound dis-played a remarkable 1000-fold enhanced binding affinityrelative to the trisaccharide Neu5AcR(2-3)Gal(1-3)Gal-NAc-2-(trimethylsilyl)ethyl. Although the compound wasnot tested in cellular regeneration assays, it was anticipatedto have improved pharmacokinetic properties due to its lowermolecular weight and favorable Clog P value.9193 Similarapproaches may yield additional therapeutic leads with thedesired inhibitory potency and pharmacokinetics for thetreatment of demyelinating disorders.

2.3.3. Synthetic Mimics of R(2-8)-Linked PSA for NerveRegeneration

PSA expression is generally considered a permissivedeterminant in areas of neuronal growth and plasticity,making it a potential therapeutic target for neuronal regen-eration. In fact, expression of PSA has been shown topromote functional recovery and provide a favorable envi-ronment for axonal regeneration in animal models of spinalcord injury.94,95 In these studies, PSA-NCAM was ectopi-cally expressed in spinal cord astrocytes in ViVo,94 or PSA-expressing Schwann cell grafts were employed.95 Althoughthe use of PSA oligo- and polysaccharides may be viablealternatives, PSA isolated from natural sources is oftenheterogeneous in length and can be contaminated with othercell-surface glycans. In addition, PSA adopts a helicalconformation96 and forms filament bundles,97 thus exhibitingdifferent structural elements that may have distinct functions.

To circumvent these challenges, Rougon, Schachner, andco-workers screened a large peptide library to identifypotential PSA mimetics.98 Two cyclic peptides were identi-fied that recapitulated the properties of endogenous PSA.Both compounds stimulated the outgrowth and defascicula-tion of mouse dorsal root ganglion (DRG) neurons andpromoted neuronal migration in Vitro and in ViVo. In addition,one peptide enhanced the migration of transplanted neuronalprogenitor cells in the murine olfactory bulb in ViVo via apathway known to be regulated by PSA.98 Thus, syntheticmimics may provide novel alternatives to PSA for neuronalregeneration.

2.3.4. Metabolic Labeling To Remodel Cell-Surface SialicAcid Interactions

The metabolic labeling of glycan chains with unnaturalsugars has played a key role in expanding the knowledge ofsialic acid function in the nervous system. Early studies byReuttar and colleagues demonstrated that unnatural chemicalfunctionalities could be incorporated into cell-surface sia-lylglycoconjugates by the addition of N-acetylmannosamineanalogues (ManNAc; Figure 5A) to cells.99103 ManNAc isthe first committed intermediate in the sialic acid biosyntheticpathway, and the enzymes in this metabolic pathway arepromiscuous for some unnatural substrates.104106 As de-scribed below, the ability to alter the structures of sialyl-

Figure 4. Structure of (A) a potent disialyl MAG inhibitor and(B) a simplified mimic of the ganglioside GQ1bR with enhancedbinding affinity to MAG relative to Neu5AcR(2-3)Gal(1-3)-GalNAc.


glycoconjugates has provided key insights into the roles ofsialic acid in neuronal migration and proliferation.

2.3.4.1. Metabolic Labeling of Neurons with ElongatedN-Acyl Derivatives of Sialic Acid. Elongated N-acyl deriva-tives of ManNAc have been incorporated into sialylglyco-conjugates of PC12 cells, oligodendrocyte progenitor cells,microglia, astrocytes, and neurons from cerebellar microex-plant cultures.101,107 In these studies, cells were treated withN-propanoylmannosamine (ManNProp), wherein the N-acetylsubstituent of Neu5Ac is replaced with a longer N-propanoylgroup (Figure 5A). ManNProp was found to stimulate theproliferation of microglia relative to cells treated with thenatural sialic acid precursor, ManNAc.107 ManNProp alsoinduced the migration of oligodendrocyte progenitor cells,the precursors to oligodendrocyte cells, which play key rolesin myelin formation and become functionally impaired inneurological diseases such as multiple sclerosis.108112Interestingly, treatment with ManNProp prolonged expressionof a sialylated ganglioside involved in cell migration, theA2B5 epitope,113 revealing a potential mechanism for itsfunctional effects.

In other studies, Reutter and co-workers investigatedwhether ManNProp modulates signaling pathways withinoligodendrocytes.114 Treatment of these cells with ManNPropand the inhibitory neurotransmitter -aminobutyric acid(GABA) induced GABA-dependent oscillations in intracel-lular calcium. Calcium is an important second messenger inthe nervous system, and calcium oscillations are believed tocontribute to a highly plastic signaling system underlyingthe communication between neurons and glia.114 Interest-ingly, ionotropic GABA receptors are modified by sialicacid,115,116 suggesting that extended N-acyl substituents mayalter the functional properties of this receptor. However,ManNProp undoubtedly perturbs the expression of multiplesialylglycoconjugates at the cell surface, and direct evidencethat altered sialylation of the GABA receptor is responsiblefor the observed response is lacking. In the future, it will beinteresting to uncover the precise molecular mechanisms bywhich these modifications to sialic acid structure elicit theireffects on intracellular signaling.

ManNProp has also been shown to promote neuronalgrowth in various contexts. For instance, ManNProp inducedthe neurite outgrowth of small rat CGN, PC12 cells, andchick DRG neurons.117,118 Moreover, treatment with Man-NProp promoted reestablishment of functional connections

in the perforant pathway, which consists of projections fromthe entorhinal cortex into the dentate gyrus of the hippoc-ampus, in coculture experiments.117 Although the particularglycoconjugates responsible for these effects were notelucidated, several cytosolic proteins implicated in neuriteoutgrowth were found to be differentially expressed afterthe ManNProp treatment, including unc-33 like phosphop-rotein (ULIP), various heat shock proteins, and 14-3-3, aprotein that associates with both GABA receptors and theR(2-3)-sialyltransferase IV.117,119,120

Bertozzi and colleagues have explored the influence ofvarious ManNAc derivatives on PSA biosynthesis. N-Butanoylmannosamine (ManNBut, Figure 5A), but notManNProp, was shown to significantly inhibit PSA expres-sion in a dose-dependent manner in the NT2 neuroblastomacell line. Moreover, both human polysialytransferases re-sponsible for PSA biosynthesis (STX and PST) displayedreduced kinetic efficiencies for transfer of ManNBut andManNPent (Figure 5A), whereas ManNProp was transferredat a rate sufficient for biosynthesis.118,121 Thus, elongationof the N-acyl side chain of sialic acid may interfere withrecognition of the growing PSA chain by polysialyltrans-ferases. However, findings by Jennings and co-workerssuggest that both ManNBut and ManNProp may be partiallyincorporated into sialylglycoconjugates, as detected by flowcytometry using a monoclonal antibody that recognizesN-propanoyl- and N-butanoyl-PSA.122,123 Consistent with aninhibitory effect on PSA biosynthesis, ManNBut blockedpolysialylation of NCAM in both chick DRG neurons118 andNT2 cells124 and decreased the outgrowth of DRG neu-rons.118 The effects on neurite outgrowth were comparableto those elicited by treatment of cells with endoneuramini-dase, an enzyme that cleaves PSA residues.

2.3.4.2. Metabolic Labeling with ManNGcPA. Metaboliclabeling of neurons with unnatural sugars has also beenexploited to alter protein recognition events at the cellsurface. Treatment of neuroblastoma-glioma hybrid cellswith the sialic acid metabolic precursor N-glycolylman-nosamine pentaacetate (ManNGcPA; Figure 5A) convertedcell-surface sialylglycoconjugates from expressing Neu5Acto expressing Neu5Gc,125 a sialic acid form that is notnormally found in humans.126 Whereas Neu5Ac sialylgly-coconjugates displayed on neuronal cells bound efficientlyto MAG, the binding of MAG to cells expressing Neu5Gcsialylglycoconjugates was significantly inhibited.127 Thesestudies demonstrate the potential of metabolic labeling toserve as a useful tool for perturbing specific glycan-proteininteractions.

2.3.4.3. Chemoselective Labeling of Sialylated Cell-Surface Glycoconjugates. The ability to incorporate un-natural sugar analogues into cell-surface glycoconjugatesallows for the introduction of reactive chemical functional-ities onto glycoproteins and glycolipids, such as ketone,azide, or alkyne groups. These functionalities allow forselective labeling of proteins with reporter groups such asaffinity tags and fluorescent dyes or for the delivery oftoxins.128131 Bertozzi and co-workers have exploited N-levulinoylmannosamine (ManLev), which contains a ketonefunctionality appended to the N-acyl side chain (Figure 5A),to label neuroblastoma cells.129 Incubation of the cells withManLev resulted in incorporation of the ketone moiety intosialylated glycans in a concentration-dependent manner.Subsequent reaction with a biotin hydrazide derivative(Figure 5B) enabled visualization of sialylglycans by fluo-

Figure 5. (A) Mannosamine derivatives used for metabolic labeling(R ) H or Ac) and (B) chemoselective labeling reaction aftertreatment of cells with ManLev (R ) biotin).


rescence microscopy, revealing their presence along the cellbody and neuronal processes.132 Although the specificsialyltransferases involved are not fully understood, ManLevwas successfully incorporated into PSA, suggesting thatR(2-8)-polysialyltransferases are capable of utilizing ketone-modified precursors for PSA synthesis.132 These studiesprovide a powerful means to modulate the structure of PSAand potentially other sialylglycans with a wide variety ofchemical groups.

2.3.4.4. Summary of Sialic Acid Metabolic Labeling.Cumulatively, studies have demonstrated that unnaturalManNAc derivatives can be exploited to manipulate thestructure of sialylated glycans on neuronal cell surfaces.These studies have revealed that subtle alterations in sialicacid structure can have striking consequences for PSAbiosynthesis and biological phenomena such as neuriteoutgrowth, cell proliferation, and migration. In the future,these versatile chemical tools could be employed forvisualization of dynamic neuronal processes in ViVo, suchas activity-dependent changes in the expression or localiza-tion of sialylglycans. The ability to engineer the glycancomposition of cell surfaces and to selectively label sialylatedglycans for imaging or other applications provides a powerfulcomplementary approach to genetics and biochemistry.

3. r-L-Fucose

3.1. Structure and BiosynthesisR-L-Fucose (6-deoxy-L-galactose; Fuc) is generally ex-

pressed as a terminal monosaccharide on N- and O-linkedglycoproteins and glycolipids. As such, it often serves as animportant molecular recognition element for proteins. Fucoseis distinct from other naturally occurring sugars because itis a deoxyhexose sugar that exists exclusively in theL-configuration (Figure 6). A structurally diverse array offucosylated glycans has been identified with fucose oftenlinked to the C-2, C-3, C-4, or C-6 positions of thepenultimate galactose in glycoconjugates or to the coreGalNAc residue of N-linked glycans.1 O-Fucosylation, thedirect modification of serine and threonine residues byfucose, has also been observed on epidermal growth factor(EGF) repeats of glycoproteins such as Notch, a proteininvolved in cell growth and differentiation.133 While fucoseis not elongated in N-linked and O-linked glycans, O-fucosecan be elongated by other sugars.1

Given the structural diversity of fucosylated glycans, it isperhaps not surprising that more than a dozen differenthuman enzymes are involved in the formation of Fuc

linkages.1 Two enzymes, FUT1 and FUT2, are dedicated tothe synthesis of FucR(1-2)Gal glycans, an epitope foundon the ABO blood group antigens134136 that has also beenimplicated in synaptic plasticity.13,137,138 A gene homologousto FUT1 and FUT2, called Sec1, contains translationalframeshifts and stop codons that interrupt potential openreading frames and thus appears to be a pseudogene.134 FUT3catalyzes the synthesis of both R(1-3)- and R(1-4)-fucosylated glycans and can transfer fucose to both Gal andGlcNAc in an oligosaccharide chain, whereas FUT4-7 formonly R(1-3)-fucosylated glycans.139,140 FUT8 and FUT9generate FucR(1-6)GlcNAc structures, with FUT8 generallycatalyzing attachment of this structure to the core asparagineresidue of N-linked oligosaccharides141 and FUT9 catalyzingits attachment to a distal GlcNAc of polylactosamine chains.142FUT10 and FUT11 are putative fucosyltransferases that arereported to synthesize R(1-3)-fucosylated glycans based onsequence homology, although no functional studies have yetbeen performed.1 Finally, POFUT1 and POFUT2, alsoknown as O-fucosyltransferase 1 and O-fucosyltransferase2, catalyze the direct fucosylation of serine and threonineresidues within epidermal growth factor repeats.143,144

3.2. Neurobiological FunctionsFucosylated glycans play important roles in various

physiological and pathological processes, including leukocyteadhesion,145,146 host-microbe interactions,147,148 and neu-ronal development.149,150 They are prevalent on the gly-colipids of erythrocytes, where they form the ABO bloodgroup antigens that distinguish specific blood types.136Aberrant expression of fucosylated glycoconjugates has beenassociated with cancer,151154 inflammation,145,155157 andneoplastic processes.158,159 For instance, the fucosylatedantigens, sialyl LewisX, sialyl LewisY, and sialyl LewisB,are up-regulated in certain cancers and have been associatedwith advanced tumor progression and poor clinical progno-sis.160163 Moreover, deficiency in fucose leads to a con-genital disorder of glycosylation type IIc in humans, alsoknown as leukocyte adhesion deficiency type II (LAD II).This disorder results in the impairment of leukocyte-vascularepithelium interactions and is characterized by immunode-ficiency, developmental abnormalities, psychomotor difficul-ties, and deficits in mental capabilities.164

Although their roles in the brain are less well understood,fucosylated glycans have been implicated in neural develop-ment, learning, and memory. Here, we will highlight aspectsof their biosynthesis and functional roles in the nervoussystem.

3.2.1. Neuronal Development

Fucose has been reported to play an important role inneural development. O-Fucosylation is essential for theactivity of Notch, a transmembrane receptor protein thatcontrols a broad range of cell-fate decisions duringdevelopment., 165169 Studies suggest that fucose modulatesNotch signaling either by inducing a conformational changein the protein or by interacting directly with Notch ligands.168Notch signaling is believed to be involved in neuronalprogenitor maintenance, and governs the cell-fate decisionbetween neuronal and glial lineages. Notch signaling mayalso contribute to the behavior of differentiated neurons andneuronal migration.170 Genetic deletion of the POFUT1 geneis embryonic lethal in mice and causes developmental defects

Figure 6. Structures of various fucose derivatives and 2-dGal.


similar to those observed upon deletion of Notch receptors,including abnormal vasculogenesis, somitogenensis, andneurogenesis.171,172 These studies demonstrate the impor-tance of fucose in proper neuronal development and implicateNotch fucosylation as an important mediator of these events.

3.2.2. Learning and Memory

Multiple studies have suggested a role for fucosylation inlearning and memory. For instance, the incorporation offucose into glycoconjugates in the brain was significantlyenhanced by task-dependent learning in both chicks andrats.173176 Rats were trained in a brightness discriminationtask, in which animals learned to enter a bright chamberwhile avoiding a dark one. Trained animals demonstratedan increase in [3H]-labeled fucose incorporation into glyco-conjugates at synapses, the specialized sites of communica-tion between neurons.175 Moreover, exogenous applicationof L-fucose or 2-fucosyllactose (Figure 6) enhanced long-term potentiation (LTP), an electrophysiological model forlearning and memory, both in ViVo and in hippocampalslices.177,178

Fucose is highly enriched at neuronal synapses,13,179,180where the majority of the fucosylated glycoconjugates existas complex N-linked structures.181 Studies indicate that theactivity of fucosyltransferases increases during synaptoge-nesis182 and upon passive-avoidance training in animals.183Moreover, the cellular machinery involved in protein gly-cosylation can be found within dendrites,184,185 raising theintriguing possibility that local protein synthesis and fuco-sylation may be occurring at synapses in response to neuronalstimulation.

Further studies have specifically implicated FucR(1-2)Gallinkages in neuronal communication processes. For instance,2-deoxy-D-galactose (2-dGal; Figure 6), which competes withnative galactose for incorporation into glycan chains and thusprevents the formation of FucR(1-2)Gal linkages,186 hasbeen shown to induce reversible amnesia in animals.138,186,187In contrast, other small molecule sugars such as 2-deoxy-D-glucose, Gal, or Glc had no effect, suggesting a uniquefunction for FucR(1-2)Gal saccharides. 2-dGal has also beenreported to interfere with the maintenance of LTP, both inVitro and in ViVo.188,189 Furthermore, a monoclonal antibodyspecific for FucR(1-2)Gal190 significantly impaired memoryformation in animals, presumably by blocking formation ofthe FucR(1-2)Gal epitope.137

3.3. Chemical Approaches for Studying L-FucoseDespite intriguing evidence linking FucR(1-2)Gal sugars

to neuronal communication and memory storage, the mo-lecular mechanisms by which these sugars exert their effectsare not well understood. Recently, however, chemical toolshave been developed that are beginning to shed light on theroles of FucR(1-2)Gal lectins and glycoproteins in the brain.

3.3.1. Deoxygalactose Analogues

Hsieh-Wilson and co-workers investigated the effects ofthe amnesic compound 2-dGal and other fucosylation inhibi-tors on cultured hippocampal neurons. Inhibition of FucR-(1-2)Gal linkages using 2-dGal led to stunted neuriteoutgrowth in young neurons lacking functional synapses(Figure 7).14 In contrast, 3-deoxy-D-galactose (3-dGal), whichinhibits fucose incorporation at the C-3 position of galactose,

had no effect on neurite growth, suggesting that specificfucose linkages are important for the neuritogenic activity.The effects of 2-dGal could be successfully rescued by theaddition of excess D-Gal to the media, suggesting that theinhibition can be reversed by the de noVo synthesis ofFucR(1-2)Gal sugars.

Interestingly, 2-dGal also exerted dramatic effects on themorphology of older neurons, even after axonal differentia-tion and synaptogenesis had begun to occur.13 Applicationof 2-dGal led to a remarkable retraction of dendrites andcollapse of synapses, whereas 6-dGal had no effect. However,D-Gal was only partially able to rescue the effects of 2-dGal,which may reflect the decreased plasticity of older neurons.Thus, fucosylated glycans and, in particular, FucR(1-2)Galglycoconjugates appear to be important for modulatingneuronal morphology and maintaining functional neuronalconnections.

To gain insight into the molecular mechanisms involved,Hsieh-Wilsonandco-workerssought to identifyFucR(1-2)Galglycoproteins in the hippocampus.13 Using a gel-based massspectrometry approach, they identified synapsins Ia and Ibas the predominant FucR(1-2)Gal glycoproteins in olderhippocampal cultures and in the adult rat brain. The synapsinsare synaptic vesicle-associated proteins that play importantroles in neurotransmitter release and synaptogenesis.191,192Fucosylation of synapsin I was found to have significanteffects on synapsin expression in neurons, protecting it fromproteolytic degradation by the calcium-activated proteasecalpain. Moreover, studies using 2-dGal and synapsin I-deficient mice showed that synapsin fucosylation contributesto the profound effects of 2-dGal on neurite outgrowth andsynapse formation. However, other unknown FucR(1-2)Galglycoproteins were also involved in the process. Thesestudies provide the first evidence that FucR(1-2)Gal gly-coproteins are directly involved in neurite outgrowth andunderscore the importance of identifying the FucR(1-2)Galproteome of the brain.

Figure 7. Inhibition of FucR(1-2)Gal linkages with 2-dGal leadsto stunted neurite outgrowth in hippocampal neurons cultured for4 days in Vitro (DIV). D-Gal is able to rescue the effects of 2-dGal.3-dGal has no effect. White bar indicates 45 m. Images courtesyof C. Gama.


3.3.2. Glycopolymers and Imaging Probes

Fucose often occupies a terminal position on glycan chains,and as such, it serves as an important molecular recognitionelement for lectins. A well-studied example is the bindingof L-selectin to the fucosylated glycan sialyl LewisX, aninteraction known to be critical for leukocyte adhesion.1 Toinvestigate whether FucR(1-2)Gal lectins exist in themammalian brain, a small molecule probe was designed andsynthesized that contained the FucR(1-2)Gal epitope and abiotin moiety for imaging potential lectin receptors in thebrain (Figure 8).14 Rat hippocampal neurons were incubatedwith the small molecule probe, and the bound probe wasvisualized on the cells using a streptavidin-dye conjugate(Figure 8). Strong fluorescent staining of the cell body andneuronal processes was observed, consistent with the pres-ence of fucose-binding lectin receptors.

To investigate whether the association of FucR(1-2)Galwith these receptors would elicit a neuronal response, Hsieh-Wilson and colleagues treated cultured neurons with poly-acrylamide-based polymers displaying multiple copies of theFucR(1-2)Gal epitope.14 The FucR(1-2)Gal polymerspromoted neurite outgrowth by more than 75%, and thepotency of the compounds was dramatically enhanced withincreasing polymer concentration or carbohydrate valency.Importantly, polymers bearing other carbohydrates moieties,such as GlcNAc, Gal, FucR(1-3)GlcNAc, or only Fuc, hadno appreciable effects, indicating that the observed neurito-genic activity was specific for FucR(1-2)Gal. Together, thesestudies provide the first evidence that FucR(1-2)Gal lectinreceptors are found in the brain, and they identify a novelcarbohydrate-mediated pathway for the regulation of neuronalgrowth. This work also highlights the power of chemicalprobes to explore the biological effects of specific glycansand their associated receptors. It will be important in thefuture to identify the lectins involved and to elucidate thespecific mechanisms and pathways leading to neuronalgrowth.

3.3.3. Metabolic Labeling Using Alkynyl or Azido FucoseAnalogues

Recently, the Bertozzi and Wong groups independentlydemonstrated that alkynyl- or azido-containing fucose ana-logues could be exploited to selectively label and imagefucosylated glycans in mammalian cells.193,194 Their strategyexploits the fucose salvage pathway to convert unnaturalfucose sugars into the corresponding GDP-fucose analogues,which then serve as donors for fucosyltransferases. Once theazido or alkynyl fucose analogue is incorporated into glycans,it can be reacted with fluorescent dyes, biotin, or peptidesvia Staudinger ligation or [3 + 2] azide-alkyne cycloaddi-tion chemistry. Bertozzi and co-workers synthesized fucosederivatives with azido groups at the C-2, C-4, and C-6positions.193 Only the C-6 azido fucose analogue (Figure 6)was successfully incorporated into the glycans of the JurkatT lymphocyte cell line, consistent with earlier observationsthat some fucosyltransferases tolerate substitutions at the C-6position of the pyranose ring. Wong and colleagues dem-onstrated that both azido- and alkynyl-modified fucosederivatives (Figure 6) could be incorporated into the glycansof hepatoma cells, allowing for fluorescent imaging offucosylated glycoconjugates.194,195 Interestingly, the alkynylfucose analogue was shown to be significantly less toxic tocells than the azido fucose analogue.194 Future applicationof these powerful approaches to neurons should facilitateproteomic studies to identify fucosylated glycoproteins andmay allow for the dynamic imaging of protein fucosylationin ViVo.

3.3.4. Summary of Fucosyl Oligosaccharides

Cumulatively, studies using chemical probes have revealeda role for fucosyl oligosaccharides and their associated lectinsand glycoproteins in the regulation of neurite growth andsynapse formation. These findings may shed light onbehavioral and electrophysiological studies implicatingFucR(1-2)Gal in long-term memory storage. Alterations inneuronal morphology, such as dynamic changes in dendriticspine number and shape, occur during memory consolidationand LTP.196,197 One possibility is that the interaction betweencertain FucR(1-2)Gal glycoproteins and lectins may promotethe stabilization of synaptic connections that underlie learningand memory. In addition, fucosylation may exert its effectsindependently of lectins, by stabilizing fucosylated glyco-proteins such as synapsin or modulating their functions. Thecontinued development and application of chemical tools hastremendous potential to expand our understanding of the rolesof fucosylated lectins and glycoproteins in the brain and mayprovide exciting opportunities to modulate neuronal com-munication processes.

4. O-GlcNAc Glycosylation

4.1. Structure and Biological FunctionsO-GlcNAc glycosylation is the covalent attachment of

-N-acetylglucosamine to serine and threonine residues ofproteins (Figure 9). Unlike other forms of glycosylation,O-GlcNAc is a dynamic, reversible modification found onlyon intracellular proteins, rendering it akin to protein phos-phorylation. A wide range of proteins are O-GlcNAc-modified, including transcription factors, nuclear pore pro-teins, cytoskeletal proteins, and synaptic proteins.8,12,198,199202

Figure 8. Chemical probe for imaging lectin receptors (top) andstaining of hippocampal neurons in culture (bottom panels) withthe probe demonstrating the presence of FucR(1-2)Gal lectins alongthe cell body and neurites. Cells were treated with 3 mM of theimaging probe (A) or biotin (B), labeled with a streptavidin-dyeconjugate, and imaged by fluorescence microscopy. Images courtesyof C. Gama.


Several excellent reviews have described the functional rolesof O-GlcNAc in transcription,203 apoptosis,204,205 signaltransduction,199 nutrient sensing,206,207 and proteasomaldegradation.206 O-GlcNAc glycosylation has also beenimplicated in the cellular stress response208,209 and is inducedby oxidative, osmotic, metabolic, and chemical stress.8,206Levels of O-GlcNAc glycosylation are altered in diseasestates such as cancer, diabetes, and Alzheimers dis-ease.201,204,207,210215 Moreover, one of the hallmarks ofAlzheimers disease is the formation of neurofibrillary tanglesby hyperphosphorylated tau protein,216 and several studiessuggest that O-GlcNAc glycosylation negatively regulatesthe ability of tau to become phosphorylated.217,218 Thus, theinvestigation of O-GlcNAc function may provide insightsinto our understanding of critical cellular processes anddiseases.

4.2. Neurobiological Functions of O-GlcNAcEmerging evidence indicates an important role for O-

GlcNAc glycosylation in the nervous system. The enzymesthat catalyze the addition and removal of O-GlcNAc,O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), aremost highly expressed in the brain219 and are enriched inboth pre- and postsynaptic nerve terminals.220 OGT expres-sion is critical for cell survival,221 and neuronal-specificdeletion of the OGT gene in mice leads to abnormaldevelopment, defects in motor coordination, and earlyneonatal death.222 Thus far, more than 50 neuronal proteinshave been shown to be O-GlcNAc-modified, includingproteins involved in transcription (e.g., CREB (cAMP-response element binding-protein), Sox2 (SRY box-contain-ing gene 2), ATF-2 (activating transcription factor-2)),neuronal signaling (synGAP (synaptic Ras GTPase activatingprotein)), bassoon, the guanine nucleotide exchange factorPDZ-GEF, and synapsin I), synaptic plasticity (synaptopodinand -catenin), and neurodegenerative disease (tau and APP(-amyloid precursor protein)).8,202,217,223227 Finally, O-GlcNAc glycosylation levels are dynamically modulated byexcitatory stimulation of the brain in ViVo and upon activationof specific kinase pathways in cultured cerebellar neurons.223

Despite its importance, the functional roles of O-GlcNAcglycosylation are only beginning to be understood in thebrain. A major challenge has been the difficulty of detectingand studying the modification in ViVo. Similar to phospho-rylation, O-GlcNAc is often dynamic, substoichiometric,targeted to subcellular compartments, and prevalent on lowabundance regulatory proteins. The sugar is also bothenzymatically and chemically labile. For example, massspectrometry analyses to identify O-GlcNAc-modified pro-teins and map glycosylation sites are challenged by loss ofthe modification upon collision-induced dissociation (CID).The lack of a well-defined consensus sequence for OGT hasprecluded the determination of in ViVo glycosylation sitesbased on primary sequence alone. Furthermore, the complex-ity of the nervous system and its unique technical challenges(e.g., postmitotic cells, multiple cell types, blood-brainbarrier, complex organization) greatly complicates efforts to

study O-GlcNAc glycosylation and necessitates the develop-ment of rapid, highly sensitive detection methods. Here, wedescribe chemical approaches undertaken to overcome thesechallenges and highlight how they have advanced ourunderstanding of the roles of O-GlcNAc glycosylation inneuronal function and dysfunction.

4.3. Chemical Tools To Study O-GlcNAcGlycosylation4.3.1. Chemoenzymatic Labeling of O-GlcNAc Proteins

4.3.1.1. Rapid, Sensitive Detection. Traditional methodsfor detecting O-GlcNAc-modified proteins often suffer fromlimited sensitivity and specificity. For instance, radiolabelingof the proteins using UDP-[3H]-galactose and (1-4)-galactosyltransferase (GalT), an enzyme that transfers [3H]-galactose onto terminal GlcNAc groups of glycoproteins,228can require weeks for visualization and lacks the sensitivityto detect certain O-GlcNAc-modified proteins. Lectins228 andantibodies229,230 are also effective methods, but they bindonly a subset of the O-GlcNAc-modified proteins (usuallythose with multiple glycosylation sites) and have limitedaffinity and specificity.

In response, a chemoenzymatic approach for taggingO-GlcNAc proteins was developed by Hsieh-Wilson and co-workers that allows for more rapid and sensitive detection.An unnatural substrate for GalT was designed, in which abioorthogonal ketone moiety was appended to the C-2position of galactose (UDP-ketogalactose probe, Figure10A).231 Studies by Qasba and colleagues had demonstratedthat a mutant form of GalT (Y289L) tolerates minorsubstitutions at this position.232 Once transferred, the ketonemoiety can be reacted with an aminooxy biotin derivative,thus permitting the sensitive detection of O-GlcNAc-modifiedproteins by chemiluminescence.231 Notably, this methodenables the identification of O-GlcNAc-glycosylated proteinsthat elude detection using other methods. For example,detection of the glycoproteins R-crystallin and CREB wasaccomplished within minutes, whereas lectins and antibodiesfailed to detect the modification on these proteins and tritiumlabeling required more than a week to develop.231 Thus, thischemoenzymatic approach provides superior sensitivity rela-tive to traditional methods and accelerates the identificationof new O-GlcNAc-modified proteins.

4.3.1.2. Identification of O-GlcNAc-Glycosylated Pro-teins from Cells. Selective biotinylation of proteins usingthe chemoenzymatic approach also facilitates the parallelpurification of O-GlcNAc-modified proteins from cell ortissue extracts by affinity chromatography.233 Previousmethods have necessitated purification of individual proteinsprior to analysis, a tedious and time-consuming process.Using the chemoenzymatic approach, the tagged O-GlcNAcproteins can be isolated in a single step by streptavidinaffinity chromatography and interrogated for modificationin parallel by Western blotting.233 This strategy was used todemonstrate that the AP-1 transcription factors c-Fos andc-Jun, as well as the activating transcription factor ATF-1,are O-GlcNAc-modified in HeLa cells.233 In addition, theidentification of O-GlcNAc on CREB-binding protein (CBP)reveals a new class of O-GlcNAc-glycosylated proteins, thehistone acetyltransferases (HAT). Thus, glycosylation canbe readily investigated across structurally or functionallyrelated proteins, as well as novel functional classes. Together,studies have revealed that a broad number of transcriptional

Figure 9. O-GlcNAc glycosylation.


components are O-GlcNAc-glycosylated,202,223,233 and O-GlcNAc may function as a general regulatory modificationfor the control of transcription.239,240

4.3.1.3. Proteome-Wide Analyses. When used in con-junction with high-throughput mass spectrometry, thechemoenzymatic approach can be exploited for proteome-wide analyses of O-GlcNAc-modified proteins.202 Proteinsfrom cell lysates are chemoenzymatically labeled and pro-teolytically digested. The desired glycopeptides are thencaptured by avidin affinity chromatography and analyzed byHPLC in line with tandem mass spectrometry (LC-MS/MS).The ketogalactose-biotin tag facilitates the isolation ofO-GlcNAc glycopeptides from complex mixtures. Thisenrichment step is often crucial for detecting low-abundancepost-translational modifications. The tag also provides aunique signature on the mass spectrometer, thus enablingunambiguous identification of O-GlcNAc-modified peptidesand mapping of glycosylation sites to specific functionaldomains within a protein. Using this approach, Hsieh-Wilson,Peters, and colleagues reported the first proteome-wideidentification of O-GlcNAc-modified proteins from themammalian brain.202 Nearly 100 peptides were identifiedcontaining the mass spectrometry signature, and 34 of thesepeptides were successfully sequenced. The sequenced pep-tides identified 25 different proteins from rat brain. Of theproteins identified, only two proteins had been previouslyreported, and 23 were novel O-GlcNAc-glycosylated pro-teins, thus significantly expanding the repertoire of proteinsknown to be modified.

This method demonstrates the power of chemical-taggingapproaches to accelerate the high-throughput identificationof O-GlcNAc glycoproteins. Notably, many of the proteinsidentified have important functional roles in gene regulation,

cytoskeletal dynamics, neuronal signaling, and synapticplasticity. For example, synaptopodin, synGap, and shank2(SH3 and multiple ankyrin repeat domains protein 2) arecritical for the regulation of dendritic spine formation.234236Synaptopodin and -catenin have important roles in learningand memory,234,237 and the guanine nucleotide exchangefactor PDZ-GEF is involved in the assembly of signaltransduction complexes at the synapse.238 Together, thesestudies suggest that O-GlcNAc glycosylation may play a rolein mediating neuronal communication and signaling net-works. Consistent with this observation, Burlingame and co-workers recently employed lectin weak-affinity chromatog-raphy in conjunction with mass spectrometry to identify 18O-GlcNAc-glycosylated proteins from the postsynaptic den-sity fraction of rat brain.224 The proteins represent multiplefunctional classes, and several proteins involved in synapticvesicle cycling were found to be extensively O-GlcNAc-glycosylated, such as bassoon, piccolo, and synapsin I.224

While the chemoenzymatic approach has broad applicationto the study of O-GlcNAc-glycosylated proteins from celland tissue extracts, O-GlcNAc proteins cannot be labeled inanimals using this method. In addition, the determination ofexact glycosylation sites is still difficult, because theketogalactose-biotin moiety can be lost upon CID in themass spectrometer. Instead, O-GlcNAc modification sites aremapped to short amino acid sequences within proteins, whichstill provides insight into the function of the modification.Despite these limitations, the chemoenzymatic labelingstrategy is so powerful for in Vitro analysis and proteomicsthat a variation of this approach is now commerciallyavailable for fluorescent labeling or biotinylation of O-GlcNAc-glycosylated proteins using [3 + 2] cycloadditionchemistry (Figure 10B).

Figure 10. (A) Chemoenzymatic approach for tagging O-GlcNAc glycosylated proteins, (B) UDP-azidogalactose probe for [3 + 2]cycloaddition chemistry using the chemoenzymatic approach, and (C) GlcNAz and biotin phosphine probe for metabolic labeling of O-GlcNAc-modified protein using the Staudinger ligation.


4.3.2. Metabolic Labeling of O-GlcNAc Proteins

4.3.2.1. IncorporationofGlcNAzintoO-GlcNAcProteins.A complementary strategy that enables tagging of O-GlcNAcylated proteins in living cells involves metabolicallylabeling the proteins with unnatural GlcNAc derivatives.Bertozzi and colleagues demonstrated that N-(2-azidoacetyl)-glucosamine (GlcNAz, Figure 10C) is processed by enzymesin the hexosamine salvage pathway, resulting in incorporationof a bioorthogonal azide functionality into O-GlcNAc-glycosylated proteins.241 The azido group can be subse-quently labeled with triarylphosphines via the Staudingerligation. Using this approach, the authors demonstratedsuccessful incorporation of GlcNAz into both nuclear andcytoplasmic proteins of cultured Jurkat T lymphocyte cells.In particular, selective labeling and detection of the nuclearpore protein p62, a known O-GlcNAc-modified protein with>10 glycosylation sites,242 was shown using a phosphine-FLAG probe. Although incomplete labeling of O-GlcNAc-glycosylated proteins limits the sensitivity of this approachrelative to the chemoenzymatic strategy described above,metabolic labeling with GlcNAz sugars can be performedin living cells and might allow for the dynamic imaging ofO-GlcNAc-glycosylated proteins in ViVo.

4.3.2.2. Proteomic Analysis by Metabolic Labeling.Although metabolic labeling has not yet been applied toneurons, it represents another powerful chemical approachfor the high-throughput identification of O-GlcNAc-modifiedproteins. Zhao and colleagues labeled O-GlcNAc proteinsin the HeLa cervical cancer cell line with GlcNAz and taggedthem with a biotin phosphine reagent (Figure 10C).243,244Tryptic digestion of the affinity-captured proteins, followedby LC-MS/MS analysis, led to the identification of 199putative O-GlcNAc-modified proteins. Because the presenceof the GlcNAc moiety was inferred rather than detecteddirectly, independent confirmation of the modification byimmunoblotting was required and demonstrated on 23 of the199 proteins.

While this method provides a powerful chemical tool forprofiling O-GlcNAc-modified proteins, there are some limi-tations of this procedure for in ViVo labeling in the brain.Most sugars do not cross the blood-brain barrier,245 andthus in ViVo labeling with these molecules would entailinvasive surgical procedures for intracranial administrationrather than simple intraperitoneal injection. In addition,metabolic labeling is not quantitative, which may limit thesensitivity of detection as well as preclude the ability tomonitor glycosylation dynamics. Despite these limitations,the approach has been successfully employed to investigatethe O-GlcNAc proteome in both mammalian and insect celllines.243,244 In the future, metabolic labeling could prove auseful tool for studying the O-GlcNAc proteome in culturedneurons.

4.3.3. Methods for Mapping Exact Glycosylation Sites

4.3.3.1. The -Elimination Followed by Michael Addi-tion with Dithiothreitol (BEMAD) Approach. The iden-tification of O-GlcNAc modification sites within proteins iscritical for elucidating the functions of O-GlcNAc in specificbiological contexts. Nonetheless, the exact sites of glycosy-lation remain unknown for most proteins. Mapping glyco-sylation sites has been challenging due to the low abundanceof the modification and the lability of the glycosidic linkageduring fragmentation on a mass spectrometer, which canresult in the loss of direct amino acid identification. Hartand co-workers showed that the labile GlcNAc moiety couldbe replaced with a more stable sulfide adduct by alkaline-induced -elimination followed by Michael addition withdithiothreitol (BEMAD, Figure 11).246 The resulting sulfideadduct is not cleaved upon CID, thereby allowing sites ofglycosylation to be more readily determined. However, alimitation of this approach is that it is often destructive toproteins,247,248 and selectivity controls must be performedto distinguish among O-GlcNAc, O-phosphate, and otherO-linked carbohydrates.246 When biotin pentylamine is usedin place of dithiothreitol, O-GlcNAc-modified peptides canbe selectively biotinylated, enriched by affinity chromatog-raphy, and identified by LC-MS/MS analysis. This methodhas been successfully employed to identify novel O-GlcNAcsites on purified glycoproteins such as synapsin I and proteinsfrom a purified rat brain nuclear pore complex.246 Furtherextension of BEMAD to complex mixtures for the high-throughput mapping of O-GlcNAc sites is an important futuregoal.

4.3.3.2. Electron Transfer Dissociation (ETD) and Elec-tron Capture Dissociation (ECD) Coupled with LectinAffinity Chromatography or Chemoenzymatic Labeling.Recently, the development of novel fragmentation methodsfor mass spectrometry has facilitated the identification ofO-GlcNAc modification sites. Electron transfer dissociation(ETD) and electron capture dissociation (ECD) use thermalelectrons to produce sequence specific-peptide fragmentationwithout the loss of labile post-translational modifications suchas O-GlcNAc and O-phosphate.249 ECD has recently beenused by Burlingame and co-workers to identify O-GlcNAcglycosylation sites following enrichment of the modifiedpeptides by lectin weak-affinity chromatography.224 Theauthors were able to identify glycosylation sites on severalneuronal proteins such as spectrin 2, shank2, bassoon, andpiccolo.

While ECD requires the use of a Fourier transform massspectrometer, ETD has the advantage of being performed inappropriately modified ion trap mass spectrometers, renderingthe technology powerful and more accessible. Hsieh-Wilson,Coon, and colleagues have implemented ETD fragmentationto map glycosylation sites on neuronal proteins following

Figure 11. BEMAD approach for mapping O-GlcNAc glycosylation sites.


chemoenzymatic labeling and enrichment by avidin affinitychromatography. The authors identified glycosylation siteson multiple proteins such as the neuron-specific transcrip-tional repressor BHC80, the transcriptional repressor p66,the transcriptional coactivator SRC-1, and the zinc fingerRNA-binding protein.223 With further methodological refine-ments and advances in database search algorithms forfragment ions, it is anticipated that ETD and ECD willbecome increasingly powerful tools for the study of O-GlcNAc glycosylation.

4.3.4. Monitoring O-GlcNAc Dynamics

Unlike most forms of protein glycosylation, O-GlcNAcglycosylation is reversible and dynamic. Several studies haveshown that global O-GlcNAc levels in cells change withinminutes of activation by specific extracellular stimuli.250,251O-GlcNAc levels are also highly responsive to cellularglucose concentrations, as approximately 2-5% of allglucose is metabolized through the hexosamine biosynthesispathway to generate UDP-GlcNAc.252254 Furthermore,studies have suggested a potential interplay between O-GlcNAc glycosylation and phosphorylation in neurons. Aninverse relationship between O-GlcNAc and O-phosphatewas observed upon activation of protein kinase C (PKC) orcAMP-dependent protein kinase (PKA) in the cytoskeletalprotein fraction of cultured cerebellar neurons.255 As de-scribed below, recent quantitative proteomics studies haveshown that O-GlcNAc glycosylation is dynamically inducedby excitatory stimulation of the mammalian brain in ViVo.223Finally, O-GlcNAc glycosylation is known to be dysregulatedin multiple disease states and is believed to contribute tothe etiology of certain diseases, such as diabetes, Alzheimersdisease, and cancer.207,252,256,257

Despite considerable investigation, the specific proteinsundergoing dynamic changes in glycosylation remain largelyunknown. Moreover, the molecular mechanisms and signal-ing pathways involved in the regulation of OGT and OGAare poorly understood. As such, there is a great need todevelop chemical tools to monitor changes in glycosylationon specific proteins and at specific modification sites in bothnormal and disease states. We describe below some of thechemical approaches that have been developed to addressthese challenges.

4.3.4.1. FRET-Based Sensors. Mahal and colleaguesdeveloped a fluorescence resonance energy transfer (FRET)-based sensor to investigate O-GlcNAc glycosylation dynam-ics in living cells.258 Their approach uses two fluorophores,enhanced cyan and yellow fluorescent protein, separated by

a known OGT substrate domain and the bacterial O-GlcNAclectin GafD (Figure 12). Upon O-GlcNAc glycosylation ofthe substrate domain, the GafD domain binds the carbohy-drate moiety, bringing the fluorophores into close proximityand leading to a concomitant increase in FRET. The authorsdetected a significant increase in FRET from HeLa cellstransfected with the sensor construct upon treatment withglucosamine or the OGA inhibitor PUGNAc (O-(2-acet-amido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcar-bamate, Figure 14). 258 This biological sensor represents apromising tool for the investigation of O-GlcNAc glycosy-lation dynamics in response to a variety of cellular stimuli.

4.3.4.2. The Quantitative Isotopic and ChemoenzymaticTagging(QUIC-Tag)ApproachforQuantitativeProteomics.Hsieh-Wilson, Peters, and co-workers developed a methodto probe dynamic changes in O-GlcNAc glycosylation usingquantitative mass spectrometry-based proteomics.223 TheirQUIC-Tag approach (quantitative isotopic and chemoenzy-matic tagging) involves chemoenzymatically labeling pro-teins from two different cell states (e.g., normal versusdiseased; stimulated versus unstimulated) with the keto-galactose-biotin group as described above (Figure 13).223After proteolytic digestion, the resulting peptides are isoto-pically labeled with either heavy or light isotope tags usingreductive amination chemistry to distinguish the two popula-tions. The peptides are subsequently combined, and thebiotinylated O-GlcNAc peptides are captured using avidinchromatography. MS analysis reveals two ions for eachglycosylated peptide (corresponding to each of the twoisotopically labeled forms), and calculation of the peak areasmeasures the change in glycosylation level for each peptide.Importantly, as the observed peptides are sequenced usingCID or ETD MS, the method identifies specific proteinsundergoing dynamic changes in glycosylation and can beused to monitor changes at particular glycosylation siteswithin proteins.

This approach has advantages over other methods ofO-GlcNAc detection. For instance, lectins and O-GlcNAcantibodies are typically used to detect only global changesin O-GlcNAc glycosylation by Western blotting and do notmonitor individual glycosylation sites. Metabolic labelingusing GlcNAz may alter the kinetic efficiency of O-GlcNActransfer to protein substrates, as well as influx through thehexosamine biosynthesis pathway, which complicates effortsto quantify dynamic changes in response to cellular stimuli.In contrast, the QUIC-Tag approach is performed ondenatured protein lysates and thus preserves the physiological

Figure 12. A fluorescence resonance energy transfer (FRET)-based sensor to detect O-GlcNAc glycosylation levels.


glycosylation state of the protein without perturbing intra-cellular glycosylation pathways.

By this approach, O-GlcNAc glycosylation was found tobe stimulated upon PUGNAc treatment of cortical neuronsor kainic acid-induced excitatory stimulation of rodent brainsin ViVo.223 Robust changes in O-GlcNAc glycosylation wereobserved at specific sites on several proteins, whereas othermodification sites remained unchanged, suggesting thatO-GlcNAc is subject to complex regulation in neurons. Forexample, glycosylation of early growth response-1 (EGR-1), a transcription factor involved in long-term memoryformation and cell survival,259,260 increased greater than 10-fold after kainic acid stimulation. Because the dynamic

glycosylation site within EGR-1 lies within its transactivationdomain, O-GlcNAc glycosylation may modulate the tran-scriptional activity of EGR-1 and modulate gene expression.Cumulatively, these studies indicate that O-GlcNAc glyco-sylation is reversible, subject to complex regulation, andinduced by neuronal activity, which supports the notion thatO-GlcNAc represents an important regulatory modificationin the brain.

4.3.4.3. Stable Isotope Labeling with Amino Acids inCellCulture(SILAC)CoupledwithAffinityChromatography.Recently, Hart and co-workers employed the SILAC (stableisotope labeling with amino acids in cell culture) methodfor quantitative proteomics261 in conjunction with immu-noaffinity chromatography to investigate the interplay be-tween O-GlcNAc and phosphorylation in COS-7 kidneyfibroblast cells.262 Cells from two different states werelabeled with either heavy or light isotopes of arginine andcombined. Proteins of interest were subsequently isolatedby affinity chromatography using a general O-GlcNAcantibody, resolved by SDS-PAGE, proteolytically digested,and analyzed by LC-MS/MS.

Using this approach, Hart and colleagues investigated theeffects of lithium inhibition of glycogen synthase kinase-3(GSK-3) on O-GlcNAc glycosylation levels. GSK-3 isinvolved in multiple intracellular signaling cascades and isimplicated in the etiology of Alzheimers disease, diabetes,and bipolar disorder, thus making it a desirable therapeutictarget.263,264 The authors identified 10 proteins that wereenriched after LiCl treatment, suggesting that they underwentincreases in O-GlcNAc glycosylation. The increases inglycosylation were confirmed on four proteins by immuno-precipitation. Interestingly, many proteins exhibited nochange, and 19 proteins showed decreases in glycosylation.These studies suggest that a complex interplay exists betweenO-phosphate and O-GlcNAc within signaling networks.

Although this approach works well for dividing cells,SILAC is not amenable to tissues and quiescent cells suchas neurons. In addition, the method does not readily enabledirect detection of the O-GlcNAc modification, and thusindependent confirmation by immunoprecipitation is required.Nonetheless, this approach provides another powerful strat-egy to investigate the cellular dynamics of O-GlcNAc glyco-sylation.

Figure 13. QUIC-Tag approach for quantifying dynamic changes in glycosylation.

Figure 14. Small-molecule OGA inhibitors.


4.3.4.4. Small-Molecule Inhibitors of OGT and OGA.Traditional genetic approaches have revealed insights intothe functions of OGT and OGA in ViVo. For example, geneticdeletion of the OGT gene in mice has revealed that OGT iscritical for cell survival, and neuron-specific deletion of OGTresults in defects in mouse embryogenesis, loss of locomotorcontrol, and neonatal death.221,222 Although such studies haverevealed an important role for these enzymes in neuraldevelopment, investigations into the functions of O-GlcNAcremain challenging, particularly in adult animals. Thedevelopment of small-molecule inhibitors for OGT and OGAhas been actively pursued to enable direct temporal andspatial control over OGT and OGA activity.

Well-known small-molecule inhibitors of OGT such asalloxan (Figure 14) show multiple nonspecific effects suchas inhibition of OGA and glucokinase,265,266 as well asformation of superoxide radicals.267 To develop betterpharmacological agents, Walker and co-workers screened alibrary using a high-throughput, fluorescence-based assay andidentified several novel compounds that inhibited OGTactivity in Vitro.268 Notably, the compounds selectivelyinhibited OGT but not MurG, a related enzyme that also usesUDP-GlcNAc as a substrate.

As PUGNAc, the most commonly used OGA inhibitor,suffers from nonspecific activity toward -hexosaminidase,269several groups are working to develop more selectiveinhibitors. The Vocadlo and Hanover groups have extendedthe N-acyl substituent of PUGNAc to generate inhibitors with10-fold selectivity for OGA over -hexosaminidase.269,270van Aalten and colleagues developed a nagstatin derivativebased the crystal structure of a bacterial OGA (Figure 14).271This molecule contains an isobutanamido group at the N8position that improves selectivity by fitting into a pocket ofthe enzyme and a phenethyl group at the C2 position thatinteracts with a solvent-exposed tryptophan from bacterialOGA. More recently, the Hanover and Vocadlo groupsindependently developed novel OGA inhibitors based on thenonspecific hexosaminidase inhibitor GlcNAc-thiazaoline, byadding fluoro, azido, or alkyl substituents (Figure 14). Theresultant inhibitors exhibited over 3000-fold selectivity forOGA over -hexosaminidase.272,273

The development of such compounds may enable theselective inhibition of OGT and OGA in cultured neurons,as well as in ViVo. The ability to perturb O-GlcNAc enzymesand glycosylation levels with small molecules should revealnew information about the functional roles of O-GlcNAcglycosylation in the nervous system, as well as facilitate theidentification of signaling pathways that regulate OGT andOGA.

5. Glycosaminoglycans

5.1. Structure and DiversityGlycosaminoglycans (GAGs) are sulfated, linear polysac-

charides that represent a central component of the extracel-lular matrix (ECM) and are involved in a myriad of biologicalfunctions, including blood coagulation,274,275 angiogene-sis,276278 tumor growth and metastasis,279281 neuriteoutgrowth,282285spinalcordinjury,286288anddevelopment.289291They are composed of repeating disaccharide units containinga hexuronic acid sugar linked to a hexosamine sugar.292,293There are several classes of GAGs (Figure 15), each of whichare distinguished by backbone composition, including heparinand heparan sulfate (HS), chondroitin sulfate (CS), dermatan

sulfate (DS), keratan sulfate (KS), and hyaluronic acid (HA).Heparin and HS contain D-glucosamine (GlcN) and eitherD-glucuronic acid (GlcA) or L-iduronic acid (IdoA) connectedby R(1-4) and (1-4) linkages. In contrast, CS polymerscontain N-acetylgalactosamine (GalNAc) instead of GlcNAcin alternating (1-3) and (1-4) linkages to GlcA, whereasDS polymers have both GlcA and IdoA linked to GalNAc.Heparin/HS and CS/DS are attached to proteins throughO-linkages to serine residues via a GlcA(1-3)Gal-(1-3)Gal(1-4)Xyl (Xyl ) xylose) tetrasaccharide linker,forming glycoconjugates known as proteoglycans.294296 KSis attached to proteoglycans through either N- or O-linkages.Hyaluronic acid is unique in that it is not protein-bound andis reportedly synthesized in the plasma membrane,296,297whereas proteoglycans are synthesized in the Golgi appa-ratus.292,293

In addition to having different backbone compositions,GAGs display remarkable structural variation through sul-fation of various hydroxyl groups along the polysaccharidebackbone (Figure 15). The sulfation patterns of GAGs areincredibly diverse, owing to the large number of potentialsulfation sites and possible combinations of differentiallysulfated disaccharides linked in tandem. For example, heparinand HS disaccharide units can be sulfated at the C-2 positionof IdoA or the C-3 and C-6 positions of GlcN. The C-2 amineof GlcN can also be acetylated, sulfated, or unmodified.Similarly, CS can be sulfated at the C-4 and C-6 positionsof GalNAc, as well as the C-2 and C-3 positions of GlcA.A simple HS disaccharide has 48 potential sulfated se-quences, yielding tetrasaccharides with over 2300 possiblesulfation sequences.

GAGs also vary in chain length from 10 to 200disaccharide units, with clusters of low and high sulfationalong the polysaccharide backbone.298 Structural studiessuggest that GAGs can adopt a variety of helical conforma-tions, such as variance in helical pitch that may depend onthe associated counterion.299,300 Further structural diversityis obtained from the conformational flexibility of the pyra-nose ring of IdoA, which exists in equilibrium between thechair and skew-boat conformations when sulfated at the C-2position.298 Thus, the combination of different sequences,charge distributions, and conformations gives rise to tre-mendous chemical and structural diversity within glycosami-noglycan chains.

Figure 15. Structures of GAG subclasses. Potential sulfation sitesare indicated in red. R ) SO3 or H; R1 ) SO3, H, or Ac; n )10-200.


5.2. Neurobiological Functions5.2.1. Neuronal Development

Evidence from genetic and biochemical approaches sug-gests that the sulfation patterns of GAGs are important formodulating their biological activity and can exert profoundeffects on organismal development. For instance, mutationof the N-deacetylase-N-sulfotransferase gene (Ndst-1) in-volved in HS biosynthesis inhibits growth factor signalingthat disrupts normal embryonic development in Droso-phila.290 HS and CS have been shown to interact withnumerous growth factors and axon guidance proteins in asulfation-specific manner.283,301308 Moreover, the sulfationpatterns of HS and CS change during the course of braindevelopment,309,310 and specific CS sulfation patterns aredifferentially expressed in certain brain regions.311,312 Thesulfation patterns of HS and CS are also organ- and age-specific, as is the expression of different sulfotransferases.309,310Thus, HS and CS sulfation patterns in the brain are tightlyregulated with the exquisite spatial and temporal controlrequired for neuronal development.

5.2.2. Axon Guidance

In the developing nervous system, neurons are presentedwith a variety of molecular cues that guide axons to theirproper targets. HS sulfation has been implicated in axontargeting through the interaction of the HS proteoglycanglypican-1 with Slit, a secreted protein important for axonguidance, axon branching, and neuronal cell migration.313,314Slit repelsaxonalgrowthbybinding to theRoboreceptor.314,315Removal of HS by heparinase treatment or addition ofexogenous HS containing specific sulfation patterns inhibitsSlit binding to Robo and abolishes the axonal repulsionmediated by Slit.304,315 These results suggest that HS andparticular HS sulfation patterns play important roles inmediating the chemotropic actions of Slit. In other studies,HS sulfation was shown to be critical for neuronal outgrowthand axon guidance in Caenorhabditis elegans. Using geneticapproaches, Hobert and colleagues demonstrated that certainneuronal subtypes require the HS-modifying enzymes C5-epimerase, 2-O-sulfotransferase, and 6-O-sulfotransferase forproper axon guidance.316 Interestingly, other subclasses ofneurons require only the C5-epimerase or 2-O-sulfotrans-ferase, and some neuronal subtypes do not require any ofthe HS modifying enzymes. Cumulatively, these studiesdemonstrate that HS sulfation patterns play important rolesin neuronal development and may encode axon guidance cuesto direct neurons to their proper targets in ViVo.

5.2.3. Spinal Cord Regeneration

Chondroitin sulfate proteoglycans (CSPGs) are crucialcomponents of perineuronal nets, structures of ECM mol-ecules surrounding the soma and proximal dendrites ofcertain neurons in the brain and spinal cord.317,318 CSPGsand other ECM molecules are recruited to sites of CNS injuryand form a portion of the glia scar, a structure that inhibitsaxonal regeneration and contributes to permanent paralysisin ViVo. Several groups have demonstrated the importanceof CSPGs and their associated sugar chains in mediatingneuronal inhibition after spinal cord injury. For instance,CSPGs have been shown to inhibit the neurite outgrowth ofDRG and CGN neurons in Vitro.319,320 Moreover, degradingCS chains with chondroitinase ABC (ChABC), an enzyme

that cleaves CS into disaccharide units, reverses the inhibitoryeffects of CSPGs on neurite outgrowth.321,322 Most notably,Fawcett, McMahon, and colleagues discovered that ChABCdigestion of CSPGs promotes spinal cord regeneration inViVo, with concomitant partial recovery of proprioceptivebehaviors and locomotor skills in mice.323,324 These and otherstudies indicate that CSPGs exert a crucial inhibitory roleon neuronal regeneration and represent valid targets fortherapeutic intervention. Such studies also underscore theimportance of CS glycosaminoglycans in this process andthe need to further understand the molecular mechanismsand sulfation patterns involved in directing their activity.

5.3. Challenges to the Study of GAGsWhile GAGs play a fundamental role in many neurobio-

logical processes, a molecular level understanding of the rolesof specific sulfation sequences in mediating GAG functionsis largely unknown. GAG biosynthesis is not template drivenand lacks the proofreading capabilities of DNA biosynthesis,which results in greater chemical heterogeneity and structuraldiversity within GAG chains. Thus, GAGs purified fromnatural sources are often mixtures of compounds that containdifferent sulfation patterns and chain lengths. Characteriza-tion of these structures is challenging and is often describedsimply in terms of the percent composition of distinct sulfateddisaccharide subunits. Little is known about the precise linearsequences of GAG polysaccharides, although methods tosequence short oligosaccharide sequences are becomingavailable.325327 Given these challenges, the synthesis ofhomogeneous oligosaccharides containing defined sulfationsequences has the potential to significantly advance ourunderstanding of the structure-activity relationships ofglycosaminoglycans. Here, we will highlight chemical ap-proaches that have helped to decipher the roles of GAGs inthe nervous system and efforts to develop GAG-basedtherapeutics for neurodegenerative diseases.

5.4. Synthetic Molecules for ProbingStructure-Activity Relationships

As described above, the sulfation patterns of GAGs areimportant for directing their neurobiological functions.Although genetic approaches have revealed crucial roles forGAGs in neural development, such experiments lead toglobal changes in sulfation throughout the carbohydratechain, precluding the identification of specific sulfation motifsresponsible for biological activity. The use of chemicallydefined small-molecule GAGs has provided insight into theirneurobiological roles and demonstrated the importance ofspecific sulfation sequences in mediating GAG functions.

5.4.1. Synthesis of Glycosaminoglycans

Early work on glycosaminoglycans focused primarily onthe synthesis of heparin oligosaccharides.328336 Heparin hasbeen used since the 1940s as an antithrombic agent, and aunique heparin pentasaccharide sequence was discovered in the1980s as a potent factor Xa inhibitor.298 The first syntheses ofheparin pentasaccharides required over 60 steps, producedheparin in relatively low yield, and were impractical for thedevelopment of synthetic drugs. Since then, the efforts ofmultiple laboratories have contributed methods that allowfor efficient syntheses of heparin, HS, and their ana-logues.337344


GAGs are notoriously difficult to synthesize, requiring theformation of stereospecific glycosidic linkages, uronic aciddonors and acceptors with low chemical reactivity, andsophisticated protecting group strategies to effect regiose-lective sulfation. Heparin, HS, and DS oligosaccharides alsonecessitate efficient syntheses of the challenging L-idopyra-nosyl sugar. The synthesis of GAGs has been summarizedin several excellent reviews (see refs 337, 344347) Recently,there has been great interest in generating libraries of sulfatedcompounds to probe the role of sulfation and identifybiologically active sulfation motifs.2,285,339,340,342,348 Ingeneral, these approaches implement modular, convergentsynthetic strategies that afford multiple sulfated structuresfrom a common disaccharide synthon and thus minimize thenumber of steps.

Other strategies have employed chemoenzymatic routesto generate defined GAG oligosaccharides. Kobayashi andco-workers have capitalized on the promiscuity of hyal-uronidase, an enzyme that normally catalyzes the hydrolysisof chondroitin in ViVo, to effect glycosidic bond formationand generate GAG polymers.349353 They were able todemonstrate the efficient polymerization of N-acetylhyalo-biuronate [GlcA(1-3)GlcNAc] and N-acetylchondrosine[GlcA(1-3)GalNAc] derivatives to form HA and nonsul-fated chondroitin, respectively, as well as unnatural cho

alzheimer sialic

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