Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic

Article Cite This: Acc. Chem. Res. 2017, 50, 2693-2705

pubs.acs.org/accounts

Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications Jie Shi Chua†,‡ and Balagurunathan Kuberan*,†,‡,§,∥ †

Department of Bioengineering, ‡Department of Medicinal Chemistry, §Department of Biology, and ∥Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, Utah 84112, United States

CONSPECTUS: Glycosaminoglycans (GAGs) are polysaccharides ubiquitously found on cell surfaces and in the extracellular matrix (ECM). They regulate numerous cellular signaling events involved in many developmental and pathophysiological processes. GAGs are composed of complex sequences of repeating disaccharide units, each of which can carry many different modifications. The tremendous structural variations account for their ability to bind many proteins and thus, for their numerous functions. Although the sequence of GAG biosynthetic events and the enzymes involved mostly were deduced a decade ago, the emergence of tissue or cell specific GAGs from a nontemplate driven process remains an enigma. Current knowledge favors the hypothesis that macromolecular assemblies of GAG biosynthetic enzymes termed “GAGOSOMEs” coordinate polymerization and fine structural modifications in the Golgi apparatus. Distinct GAG structures arise from the differential channeling of substrates through the Golgi apparatus to various GAGOSOMEs. As GAGs perform multiple regulatory roles, it is of great interest to develop molecular strategies to selectively interfere with GAG biosynthesis for therapeutic applications. In this Account, we assess our present knowledge on GAG biosynthesis, the manipulation of GAG biosynthesis using synthetic xylosides, and the unrealized potential of these xylosides in various biomedical applications. Synthetic xylosides are small molecules consisting of a xylose attached to an aglycone group, and they compete with endogenous proteins for precursors and biosynthetic enzymes to assemble GAGs. This competition reduces endogenous proteoglycan-bound GAGs while increasing xyloside-bound free GAGs, mostly chondroitin sulfate (CS) and less heparan sulfate (HS), resulting in a variety of biological consequences. To date, hundreds of xylosides have been published and the importance of the aglycone group in determining the structure of the primed GAG chains is well established. However, the structure−activity relationship has long been cryptic. Nonetheless, xylosides have been designed to increase HS priming, modified to inhibit endogenous GAG production without priming, and engineered to be more biologically relevant. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. They are small, orally bioavailable, easily excreted, and utilize the host cell biosynthetic machinery to assemble GAGs that are likely nonimmunogenic. Various xylosides have been shown, in different biological systems, to have anticoagulant effects, selectively kill tumor cells, abrogate angiogenic and metastatic pathways, promote angiogenesis and neuronal growth, and affect embryonic development. However, most of these studies utilized the commercially available one or two β-D-xylosides and focused on the impact of endogenous proteoglycan-bound GAG inhibition on biological activity. Nevertheless, the manipulation of cell behavior as a result of stabilizing growth factor signaling with xyloside-primed GAGs is also reckonable but underexplored. Recent advances in the use of molecular modeling and docking simulations to understand the structure−activity relationships of xylosides have opened up the possibility of a more rational aglycone design to achieve a desirable biological outcome through selective priming and inhibitory activities. We envision these advances will encourage more researchers to explore these fascinating xylosides, harness the GAG biosynthetic machinery for a wider range of biomedical applications, and accelerate the successful transition of xyloside-based therapeutics from bench to bedside.

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INTRODUCTION

Extracellular events, including cell−cell and cell−extracellular matrix (ECM) interactions, are immensely important in dictating © 2017 American Chemical Society

Received: June 9, 2017 Published: October 23, 2017 2693

DOI: 10.1021/acs.accounts.7b00289 Acc. Chem. Res. 2017, 50, 2693−2705

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Accounts of Chemical Research

Figure 1. (A) Disaccharide structures of glycosaminoglycans. (B) Biosynthesis of HSGAGs and CSGAGs.

glycosaminoglycans (GAGs), linear polysaccharides found ubiquitously on cell surfaces and in the ECM. They regulate numerous developmental and pathophysiological events such as

intracellular signaling, gene regulation, and cell behavior, which in turn, determine the organization of cells into tissues, organs, and an entire organism. Cardinal players in these interactions are 2694


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sulfate (DS) (CSGAGs), hyaluronic acid, and keratan sulfate (Figure 1A). All but hyaluronic acid can be sulfated at various positions, resulting in a large number of possible disaccharide units, which are organized into more complex sequences. The location, size, backbone structure, and epimerization and sulfation pattern of these GAG chains confer an enormous structural diversity that accounts for their binding to numerous proteins and, thus, functional significance in many biological processes. However, with the exception of the antithrombin binding HS,1,2 no other GAG structures have been exploited clinically. Challenges, such as chemical microheterogeneity, lack of sensitive analytical tools, and structural and functional redundancy, hamper further developments. It is intriguing to note the emergence of tissue-specific GAG structures from a nontemplate driven biosynthesis that appears to lack proofreading machinery. With a focus on HS and CSGAGs, this Account outlines our current knowledge on GAG biosynthesis, the manipulation of GAG expression in biological systems using synthetic xylosides, and the potential biomedical applications of xylosides.

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GAG BIOSYNTHESIS AND THE GAGOSOME MODEL The biosynthesis of HS and CSGAGs has been extensively reviewed.3−5 It begins with the addition of a xylose to specific serine residues of the proteoglycan core proteins in the late endoplasmic reticulum (ER) or the cis Golgi apparatus (GA). Then, glycosyl residues, two galactose and one glucuronic acid (GlcA), are added sequentially by galactosyltransferases, GalT-I and GalT-II, and glucuronyltransferase 1 (GlcAT-1), respectively, forming the tetrasaccharide linker (Figure 1B). The tetrasaccharide linker undergoes further modifications that affect subsequent biosynthetic events. The xylose can undergo phosphorylation at the C2 position whereas the galactose residues can undergo sulfation at either the C4 or C6 position or both. The fifth residue added determines the GAG type: the incorporation of N-acetylgalactosamine (GalNAc) makes CSGAGs, whereas N-acetylglucosamine (GlcNAc) makes HSGAGs. Polymerization of the HS backbone proceeds with the alternate addition of GlcA and GlcNAc by an enzyme complex encoded by EXT1 and EXT2. The elongated chain then undergoes N-sulfation, C5-epimerization, and O-sulfation at specific positions, conferring binding specificity and functionality. 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the sulfate donor and is transported into the GA via PAPS transporters. As GAG structures, in terms of size, type, and epimerization and sulfation pattern, determine their biological actions, biosynthesis needs to be tightly regulated for appropriate cell- and tissue-specific responses. The rate limiting step of GAG biosynthesis is the transfer of xylose to serine and is catalyzed by the chain-initiating enzyme xylosyltransferase.6,7 In mammals, there are two xylosyltransferases, XylT1 and XylT2, and their activity and expression differ across cell and tissue types.7,8 The occurrence of xylosylation and the type of GAG formed are determined by the amino acid sequence flanking the serine. CS assembly occurs when the sequence consists of acidic residues followed by Ser-Gly-XaaGly9,10 whereas HS formation requires repetitive Ser-Gly residues flanked by a cluster of acidic residues, the substitution or truncation of which leads to reduced HS and increased CS assembly.11,12 Apart from the amino acids immediately adjacent to the serine, sequences further away from the assembly site also assert influence. For example, the globular domain of glypican-1, a major HS proteoglycan (HSPG), plays an important role for

Figure 2. (A) The GAGOSOME model of GAG biosynthesis is supported by the differential priming of GAGs by xylosides with varying aglycones. (B) Assembly of multiple GAG chains can proceed in either a parallel or sequential manner that could be deciphered with bisxylosides.

organogenesis, neuronal growth and guidance, angiogenesis, tissue homeostasis, and microbial pathogenesis, among others. There are four main classes of GAGs: heparan sulfate (HS) and heparin (HSGAGs), chondroitin sulfate (CS) and dermatan 2695


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Figure 3. (A) Endogenous GAGs are attached to proteoglycans on the cell surface. (B) Xylosides inhibit GAG assembly on endogenous proteoglycans and xyloside-primed GAGs compete with endogenous GAGs for binding proteins.

segregation occurs earlier.20 In polarized epithelial cells, proteoglycans secreted to the apical and basolateral membranes have different GAG structures, in terms of chain length, sulfation levels, and patterns.21 Increasing PAPS availability via overexpressing PAPS transporters increases sulfation of apical but not basolateral CS proteoglycans (CSPGs), while the silencing of PAPS transporters altered basolateral but not apical HS sulfation, suggesting differential regulation and divergence of the secretory pathways in the GA.22,23 Nonetheless, no direct evidence for the GAGOSOME model and interactions among all the biosynthetic enzymes exist, and this remains a topic for investigation and debate.

preferential HS assembly, and the effect extends to core proteins to which it is not physically attached, suggesting that the domain may inhibit components of the CS biosynthetic pathway.13 Although the sequence of biosynthetic events and the specificity of enzymes involved are known, the codification of distinctive cell-specific GAG structures remains enigmatic.14,15 Current understanding inclines toward the hypothesis that macromolecular complexes of GAG biosynthetic enzymes, termed “GAGOSOMEs”, coordinate polymerization and modifications in the GA (Figure 2).14−16 The differential channeling of substrates to various GAGOSOMEs generates distinct GAG structures, and the process is regulated in a stochastic manner by the amount of enzymes and substrates and their affinity. Several pieces of evidence support the GAGOSOME hypothesis. In vivo, EXT1 and EXT2 work in synergy by forming a heterooligomeric complex, which has significantly better activity than either alone.17 EXT2 also interacts with N-deacetylase-Nsulfotransferase-1 (NDST1), and HS sulfation changes with the differential expressions of EXT1, EXT2, and NDST1, possibly due to the GAGOSOME compositions that vary with the relative concentrations of these enzymes.18 2-O-Sulfotransferase prefers substrates with iduronyl residues but avoids 6-O-sulfated regions, suggesting a deliberate choreography among enzymes.19 In addition to interactions among enzymes and their substrate preferences, the GAGOSOME hypothesis further entails that differential GAG assembly arises from the different routes taken by the protein through the GA (Figure 2A). Contrary to the conventional view that the trans-GA is the major protein sorting site, increasing experimental evidence supports that lateral

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XYLOSIDES FOR PROBING AND PERTURBING GAG BIOSYNTHESIS Given the numerous roles of GAGs in biology, it is extremely useful to develop molecular tools that selectively interfere with GAG biosynthesis and, thus, GAG structures. Such approaches can aid us in the comprehension of GAG structure−function relationships and their contributions to biological processes. Synthetic xylosides, consisting of a xylose attached to an aglycone, are particularly suited for this purpose and their chemical synthesis has been extensively reviewed.24 Early studies reported that D-xylose and β-D-xylosides to act as artificial initiators of GAG biosynthesis and prime protein-free CSGAGs in cultured cells.25,26 Their effects are twofold: they act as decoys by competing with core proteins for biosynthetic enzymes to reduce endogenous proteoglycan-bound GAG assembly and they prime GAG chains that compete with endogenous proteoglycan-bound GAGs for binding to proteins, such as 2696


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biosynthesis site.28,29 Nonpolar groups were better primers, as they cross the cell membrane more efficiently to the ER and GA.28 Because the protein and lipid compositions of the plasma membrane change across cells and subcellular compartments, the desired proportion of hydrophobic moieties and, thus, optimal size of an alkyl chain as an aglycone were cell- and tissuespecific.29 GAG priming also increased with the spacer length between the xylose and the aglycone, suggesting that increased surface area makes xylosides more accessible to enzymes.30 However, other spacer properties also affect primed structures and their biological activities with no defining trends, implying the involvement of other factors such as conformational flexibility, steric hindrance, and enzyme affinity.31 Xylosides prime predominantly CSGAGs and limited HSGAGs, although HS and CSGAGs share the same tetrasaccharide linkage.29 The influence of the aglycone on HS/CS composition was highlighted when β-D-xylosides carrying two fused aromatic rings were demonstrated to prime up to 50% HSGAGs.32 There are two possible explanations: First, CS synthesis occurs by default and HS only forms when the aglycone has a similar three-dimensional structure to a domain on HSPG core proteins that GlcNAc transferase recognizes for the commitment to HS biosynthesis.13,32 A second proposition, consistent with the GAGOSOME model, is that the aglycone group facilitates the selective transport of xylosides to specific GA subcompartments where HS synthesizing enzymes are localized.31 Likewise, the dependence of xyloside transport on the aglycone and the cell type was further implied when hexyl-β-Dxyloside-primed GAGs of Madin−Darby canine kidney (MDCK) cells were demonstrated to secrete apically whereas benzyl-β-D-xyloside-primed GAGs of CaCo-2 cells secreted basolaterally.33 Although the importance of the aglycone is well established, little is known about the relationship between the aglycone structure and the primed GAGs. In an attempt to bridge this knowledge gap, our group generated a large xyloside library using click-chemistry.31 For the first time, we demonstrated that the aglycone, in terms of aromatic ring type, substituents, substituent positions, and spacer properties, affects the amount, sulfation pattern and levels, chain length, disaccharide composition, and CS/HS ratio of the primed GAGs (Figure 4).31 Concurring with the GAGOSOME hypothesis, we postulated that the structural variations arise from the differential transport of the xylosides, facilitated by the aglycone groups, to different GA subcompartments (Figure 2A). Moreover, priming activity is likely dependent on both xyloside transport and the affinity of the aglycone to the enzymes involved in forming the tetrasaccharide linker, as substrate inhibition at high concentrations was observed for some click-xylosides.31 The exceptional metabolic stability of click-xylosides makes them ideal for biological applications. To exploit this advantage, we have synthesized, using click-chemistry, cluster-xylosides that mimic endogenous multivalent proteoglycans34 and xylosides conjugated to BODIPY,35 biotin,36 and RGD.37 Clusterxylosides are excellent for studying the assembly of multiple GAG chains and the biological significance of GAG multivalency (Figure 5A). For instance, bis-xylosides always prime bivalent chains, suggesting that coordinated concurrent extension from both xylose residues likely occurs (Figure 2B).34 BODIPY- and biotin-conjugated xylosides are novel tools for tracking GAG biosynthesis and distribution. BODIPY-xyloside, for example, was used to visualize the distribution of the fluorescent GAGs over time in the inner ear of the oyster toadfish (Figure 5B).35

Figure 4. Aglycone moiety, in terms of aromatic ring type, substituents, substituent positions, and spacer properties, affects (A) chain length, (B) sulfation level, and (C) disaccharide composition of the primed GAGs.

growth factors and chemokines, with varying biological consequences (Figure 3).27 To date, hundreds of xylosides with different aglycones have been synthesized and tested for their decoy and priming effects. Xyloside activity is dependent on both the nature of the aglycone and the tissue or cell type and limited by its transport to the 2697


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Figure 5. Applications of click-xylosides in animal models. (A) Multivalent bis-xylosides upregulated FGF signaling in zebrafish embryos. (B) Biotinand BODIPY-conjugated xylosides were used to track primed GAG chains in different biological systems. (C) Various click-xylosides promote therapeutic angiogenesis in an aglycone dependent manner.

Targeting specific cells, such as tumor and activated endothelial cells, can be achieved with RGD-xylosides that also prime GAGs (Figure 6A).37 Inhibiting GAG biosynthesis through the decoy effect of xylosides can be particularly beneficial for diseases wherein GAG production contributes to pathology (Figure 6B).38 In collaboration with the Koketsu’s lab, we eliminated priming and enhanced inhibitory activities by replacing the hydroxyl group at the C4 position of xylose with fluorine. Using clickchemistry, we synthesized a repertoire of 4-deoxy-4-fluoroxylosides, the first known specific HS and CSGAG inhibitors. Depending on the aglycone and concentration, inhibitory activities ranged from 0% to 90%, demonstrating that the inhibition efficiency can be fine-tuned.38

Until recently, xyloside screening was limited to cell-based assays and the relationship between xyloside structures, priming and decoy activities, and their biological effects could only be speculated. This, however, has been transformed with the publication of human GalT1 substrate−enzyme crystal structure and the successful cloning and expression of the enzyme.39 By combining molecular docking simulations, cell-free recombinant enzyme assays, and traditional cell-based assays, it is now possible to investigate the structure−activity relationship of xylosides through their interactions with the GalT1 active site, deriving considerable insights unattainable before.39−41 These studies revealed GalT1 to have a narrow and specific binding pocket, stipulating that inhibitor design should focus on modifications at the C4 position, as other modifications are less tolerated.41 On the contrary, the aglycone can be 2698



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BIOMEDICAL APPLICATIONS OF SYNTHETIC XYLOSIDES As GAGs regulate many developmental, physiological, and pathological processes, the perturbation of GAG biosynthesis with xylosides holds great promise to ameliorate many diseases. Xylosides, however, have not been used for clinical therapy. In the following sections, potential biomedical applications of xylosides are described. Anticoagulation Therapy

The only GAG−protein interaction that has been exploited for clinical use is a specific heparin pentasaccharide that binds to antithrombin and inhibits thrombin, a critical player in the coagulation cascade (Figure 7).1,2 Thrombin is also inhibited by

Figure 6. Applications of click-xylosides in cell culture models. (A) RGD-conjugated xylosides may be used to target cells expressing αvβ3 integrin. (B) Fluoro-xylosides inhibit endogenous GAG biosynthesis and block tumor-associated angiogenesis. (C) Various clickxylosides mitigate glioma cell invasion and migration.

Figure 7. Odiparcil increases circulating anticoagulant DS, which binds to heparin cofactor II and inhibits thrombin.

larger, as it extends outside the active site.41 Xyloside-based inhibitors were designed, by Fournel-Gigleux and Ouzzine, to bind tightly to GalT1, predicted from a detailed analysis of the amino acids at and near the active site of the enzyme.40 The expanding knowledge on the activity and specificity of xylosides reveals the opportunity to rationally design aglycones for desired biological outcomes.

DS polysaccharides through the heparin cofactor II pathway.42 Clinically used heparin- and DS based blood thinners are highly complex mixtures isolated from over 700 million pigs. Conversely, xylosides prime specific GAGs with defined compositions that can potentially serve as anticoagulants. In fact, Odiparcil, an orally bioavailable β-D-thioxyloside that mostly primes 2699


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Figure 8. Xylosides have great potential to be developed as anticancer drugs, as they were reported (A) to be tumor cell specific and reduce cell surface GAGs and, thereby, to inhibit (B) proangiogenic and (C) prometastatic signaling pathways and (D) to reduce oncogenic exosome uptake.

substituents on the naphthyl ring (Figure 8A).47 Interestingly, naphthyl-xyloside-primed CS, rather than HS, was found to cause cytotoxicity only when primed in cancer cells in vitro, suggesting complex confounding mechanisms.48 In other studies, xylosides interrupted HS-mediated chemokine signaling, required for initiating metastasis, through their decoy effects that reduce cell surface proteoglycan-bound GAGs (Figure 8C).49 Likewise, xylosides were found to attenuate glioma and HeLa cell invasion in vitro (Figure 6C).49,50 Cell surface HSPGs are furthermore required for the uptake of exosomes, which carry promoters of metastatic pathways to secondary sites and maintain the cancer microenvironment by promoting proliferation, angiogenesis, and drug resistance (Figure 8D).51 β-D-Xylosides

DS, significantly reduced venous thromboembolism in patients who underwent total hip or knee replacement in phase II clinical trials.42−45 With an increased understanding of the aglycone role, greater efforts can now be directed toward designing xylosides that preferentially prime more potent GAG anticoagulants. Cancer Therapy

The hallmarks of cancer include deregulated proliferation, metastasis, and sustained angiogenesis, processes in which GAGs have been implicated and, therefore, can be prospective treatment targets.46 Inhibition of tumor cell growth by naphthylxylosides both in vivo and in vitro was attributed to their HS priming activity, an effect sensitive to different hydroxyl 2700


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Figure 9. Xylosides can contribute to the field of regenerative medicine as they have been reported to promote angiogenesis, which is important for healing and tissue survival, and they affect stem cell differentiation.

Potential applications in epithelial repair were demonstrated by C-xylosides that improve the dermal-epidermal junction and promote keratinocyte migration.55,56 Click-xyloside-primed GAGs promote and stabilize angiogenesis,57 which can benefit wound healing and tissue engineering applications wherein a vascular network is necessary (Figure 5C). Treatment of lung fibroblasts with 4-MU-4-deoxy-β-D-xyloside have antifibrotic effects attributed to the inhibition of proteoglycan synthesis,58 and this strategy can be extended to other tissues where excessive ECM deposition impedes healing. Potentially, xylosides can also be used to treat osteoarthritis, attributed to the pathological reduction of sulfated CSGAGs, and accelerate cartilage repair that has only been accomplished with increased proteoglycan synthesis through genetic approaches thus far.59 The conditions following injury to the central nervous system (CNS) are notoriously inhibitory for axonal regeneration due to

reduced exosome uptake and, correspondingly, glioma cell migration.51 Similarly, reduction of endogenous GAGs with fluoro-xylosides, specific inhibitors of HS/CSPG biosynthesis, abolished tumor-associated angiogenesis (Figure 6B).52 Angiogenesis is majorly dependent on Vascular Endothelial Growth Factor (VEGF) and FGF2 signaling, both of which require GAG binding (Figure 8B).53 Current antiangiogenic therapies, which typically act on the VEGF receptors, are unable to produce an enduring response, as cancer cells upregulate multiple proangiogenic pathways.54 Therefore, the unique ability of xylosides to concurrently abrogate many angiogenic and metastatic signaling pathways through the overall reduction of cell surface GAGs can be a more favorable therapeutic strategy. Regenerative Therapy

Xylosides can be employed to modulate cell surface and ECM GAG profiles and, in turn, direct cell behavior for regeneration (Figure 9). 2701


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Accounts of Chemical Research the deposition of CSPGs at the injury site by reactive astrocytes.60 Blocking the overproduction of CSPGs with β-D-xylosides, through their decoy effects, promoted axonal growth in cell culture models61 and functional recovery in animal models of spinal cord injury (Figure 10).60,62 On the other hand, the priming activity of

were reported to stimulate hematopoiesis of bone marrow cultures,72 regulate chondrogenic differentiation of mesenchymal stem cells,73 inhibit proliferation of neural stem cells,74 and reduce aggregation of developing chick neurons.75 Unlike the above-mentioned studies that only exploited the decoy effect of xylosides, the generation of unique GAG structures via the priming activity to regulate growth factor signaling is less investigated though desirable (Figure 9).63,64 The FGF family, comprising 22 members, is known to regulate many developmental processes. HS binding provides a storage reservoir of FGF in the ECM and facilitates FGF receptor dimerization, required for downstream signal transduction.76 An important mode of regulation is the requirement of distinct HS structures for different FGF−FGFR pairs.77 The facilitation of FGF−FGFR complex formation with bivalent GAGs, primed by bis-xylosides, upregulated FGF8 signaling, accelerating zebrafish embryo elongation (Figure 5A).36 This phenomenon was not observed with mono-xylosides that prime monovalent GAG chains, demonstrating the exciting possibility of manipulating cell signaling with bis-xyloside-primed GAGs at different developmental stages and, in turn, stem cell fate and behavior for regenerative medicine.

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CONCLUSIONS AND PERSPECTIVES Undoubtedly, xylosides are exceptionally useful for studying the role of GAGs in biological systems and promising drug candidates for diseases wherein GAGs are implicated. Being small molecules, xylosides have high diffusivity, can be easily excreted, and are unlikely to be immune reactive. An easy delivery mode was further highlighted with the effective oral administration of xylosides in animal models of thrombosis.42,43 Xylosides are advantageous over exogenous synthetic or animalderived GAGs for therapy because xyloside-primed GAGs are derived from the host biosynthetic machinery and, thus, are unlikely to invoke undesirable immune responses. When coupled with carefully designed delivery approaches, xylosides can be powerful therapeutics to alter GAG compositions at specific disease sites.37 Although the rules that govern the relationship between xyloside structures, their priming activities, and the resulting biological outcomes have remained cryptic, untangling these challenges can offer new opportunities for engineering the ECM. In fact, we have demonstrated, with our click-xyloside library, that the dependency of the primed GAG chains on the xyloside structure can be elucidated to some extent with the detailed analysis of the primed GAGs. The post-GA fate of proteoglycans is affected by information encoded in GAGs, which are affected by the aglycone, when primed by xylosides.3,21,33 Foreseeably, we can engineer an aglycone for preferentially priming and delivering anticoagulant GAGs to the luminal side of endothelial cells and another for priming GAGs that can modulate cytokine activity on the abluminal side. To date, hundreds of xylosides have been published, and researchers need to utilize them instead of relying on a very few commercially available β-D-xylosides. Furthermore, investigations with xylosides should look beyond their decoy activities and exploit their priming activities for directing cell signaling and behavior. The recent advances in molecular modeling enable the rational design of xylosides that will open up new opportunities for harnessing selectively either their decoy or priming activities and will lead to fruitful biomedical applications in this unappreciated and underexplored research frontier.

Figure 10. Xylosides are beneficial for CNS regeneration, as they support a growth-promoting environment.

xylosides also promotes neuronal growth in both cell and organotypic cultures.63,64 One can predict that the observed neuronal growth may require specific sulfation topology on the primed GAGs as was the case with defined synthetic oligosaccharides.65 Inhibitory CSPGs are also major components of the proteoglycan rich perineuronal nets (PNN), limiting neuronal growth and plasticity in the adult CNS.66 Manipulation of PNN GAG profiles with xylosides offers a hope for enhancing neuronal plasticity and functional recovery in disorders such as Alzheimer’s disease, epilepsy, and schizophrenia, for which no treatment currently exists.66 GAGs are known to regulate morphogenetic and differentiation events during development; thereupon, xylosides, as expected, caused abnormalities in the development of chick67 and rat embryos68 and inhibited the branching morphogenesis of salivary glands,69 embryonic kidneys,70 and lungs.71 Additionally, β-D-xylosides have divergent effects on different stem cells: they 2702


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Balagurunathan Kuberan: 0000-0001-9203-8118 Notes

The authors declare no competing financial interest. Biographies Jie Shi Chua received her B. Eng. with honors in Bioengineering from the National University of Singapore (2013). She is currently a graduate student in the University of Utah Biomedical engineering Ph.D. program, under the supervision of Prof. Kuberan Balagurunathan. Her research interest lies in the reengineering of the ECM for tissue engineering and regenerative medicine. Kuberan (Kuby) Balagurunathan received his Ph.D. with Prof. Robert Linhardt at the University of Iowa and subsequently conducted postdoctoral research with Prof. Robert Rosenberg at MIT before joining the faculty at University of Utah. The Kuby lab studies glycosaminoglycan biosynthesis and functions.

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ACKNOWLEDGMENTS B.K. acknowledges the contribution of all of his current and former lab members to the work described herein. The Kuby lab is supported by the NHLBI sponsored Programs of Excellence in Glycosciences grant (P01-HL107152). We thank Prof. Gary Schoenwolf for proofreading the manuscript.

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ABBREVIATIONS ECM, extracellular matrix; GAGs, glycosaminoglycans; HS, heparan sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; ER, endoplasmic reticulum; GA, Golgi apparatus; PG, proteoglycan

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REFERENCES

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Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic

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