Balagurunathan Kuberan

Enzymatic synthesis of antithrombin III–binding heparan sulfate pentasaccharide

Heparan sulfate (HS) proteoglycans are crucial to numerous biological processes and pathological conditions, but to date only a few HS structures have been synthesized and characterized with regard to structure-function relationships. Because HS proteoglycans are highly diverse in structure, there are substantial limitations on their synthesis by classical chemical means, and thus new methods to rapidly assemble bioactive HS structures are needed. Here we report the biosynthesis of bioactive HS oligosaccharides using an engineered set of cloned enzymes that mimics the Golgi apparatus in vitro. We rapidly and efficiently assembled the antithrombin III–binding pentasaccharide in just 6 steps, in contrast to the approximately 60 steps needed for its chemical synthesis, with an overall yield at least twofold greater and a completion time at least 100 times faster than for the chemical process.

Investigating the elusive mechanism of glycosaminoglycan biosynthesis.

Glycosaminoglycan (GAG) biosynthesis requires numerous biosynthetic enzymes and activated sulfate and sugar donors. Although the sequence of biosynthetic events is resolved using reconstituted systems, little is known about the emergence of cell-specific GAG chains (heparan sulfate, chondroitin sulfate, and dermatan sulfate) with distinct sulfation patterns. We have utilized a library of click-xylosides that have various aglycones to decipher the mechanism of GAG biosynthesis in a cellular system. Earlier studies have shown that both the concentration of the primers and the structure of the aglycone moieties can affect the composition of the newly synthesized GAG chains. However, it is largely unknown whether structural features of aglycone affect the extent of sulfation, sulfation pattern, disaccharide composition, and chain length of GAG chains. In this study, we show that aglycones can switch not only the type of GAG chains, but also their fine structures. Our findings provide suggestive evidence for the presence of GAGOSOMES that have different combinations of enzymes and their isoforms regulating the synthesis of cell-specific combinatorial structures. We surmise that click-xylosides are differentially recognized by the GAGOSOMES to generate distinct GAG structures as observed in this study. These novel click-xylosides offer new avenues to profile the cell-specific GAG chains, elucidate the mechanism of GAG biosynthesis, and to decipher the biological actions of GAG chains in model organisms.

Chemical Tumor Biology of Heparan Sulfate Proteoglycans

Heparan sulfate proteoglycans (HSPGs) play vital roles in every step of tumor progression allowing cancer cells to proliferate, escape from immune response, invade neighboring tissues, and metastasize to distal sites away from the primary site. Several cancers including breast, lung, brain, pancreatic, skin, and colorectal cancers show aberrant modulation of several key HS biosynthetic enzymes such as 3-O Sulfotransferase and 6-O Sulfotransferase, and also catabolic enzymes such as HSulf-1, HSulf-2 and heparanase. The resulting tumor specific HS fine structures assist cancer cells to breakdown ECM to spread, misregulate signaling pathways to facilitate their proliferation, promote angiogenesis to receive nutrients, and protect themselves against natural killer cells. This review focuses on the changes in the expression of HS biosynthetic and catabolic enzymes in several cancers, the resulting changes in HS fine structures, and the effects of these tumor specific HS signatures on promoting invasion, proliferation, and metastasis. It is possible to retard tumor progression by modulating the deregulated biosynthetic and catabolic pathways of HS chains through novel chemical biology approaches.

A new strategy for defining critical functional groups on heparan sulfate

Heparan sulfate (HS) is a sulfated polysaccharide present on cell surfaces and in the extracellular matrix. Accumulating evidence shows that HS plays key roles in many biological systems by interacting with various proteins in a structural‐specific manner. Due to technical difficulties, however, the understanding of critical functional groups on HS for protein interaction is vague. We report a rapid, convenient, sensitive, and inexpensive strategy using in vitro modification with pure enzymes and gel mobility shift assay to study the subject. We demonstrated the requirements of 3‐O,6‐O sulfates and the minimal length of oligosaccharide for antithrombin III (AT‐III) binding. We regenerated the binding sites for AT‐III on completely desulfated N‐resulfated heparin and revealed the critical modification enzymes. This new strategy could be used to identify critical functional groups on HS and to generate HS library and biologically active HS, providing information applicable to the design of HS drugs, such as anticoagulant reagents and viral infection blockers. The binding assay with fibroblast growth factors and receptors confirmed the general usefulness of this approach.—Wu, Z. L., Zhang, L., Beeler, D. L., Kuberan, B., Rosenberg, R. D. A new strategy for defining critical functional groups on heparan sulfate. FASEB J. 16, 539–545 (2002)

Chemoenzymatic Synthesis of Classical and Non-classical Anticoagulant Heparan Sulfate Polysaccharides

Heparan sulfate (HS) polysaccharides interact with numerous proteins at the cell surface and orchestrate many different biological functions. Though many functions of HS are well established, only a few specific structures can be attributed to HS functions. The extreme diversity of HS makes chemical synthesis of specific bioactive HS structures a cumbersome and tedious undertaking that requires laborious and careful functional group manipulations. Now that many of the enzymes involved in HS biosynthesis are characterized, we show in this study how one can rapidly and easily assemble bioactive HS structures with a set of cloned enzymes. We have demonstrated the feasibility of this new approach to rapidly assemble antithrombin III-binding classical and non-classical anticoagulant polysaccharide structures for the first time.

Click Xylosides Initiate Glycosaminoglycan Biosynthesis in a Mammalian Cell Line

Proteoglycans are composed of a core protein and several complex glycosaminoglycan (GAG) polysaccharide side chains. Heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS) belong to the family of GAGs. In humans, these GAG side chains have been shown to regulate many biological functions, including wound healing, cell signaling, cell differentiation, angiogenesis, blood clotting, and tumor-cell migration. GAGs consist of repeating disaccharide units of hexosamine and uronic acid, and are covalently ACHTUNGTRENNUNGattached to a serine residue of the core protein via a specific linkage tetrasaccharide (Figure 1). The very first step in GAG synthesis is xylosylation of a serine residue of the core protein, followed by assembly of a tetrasaccharide unit that serves as an acceptor for elongation of GAG chains.

Inhibition of Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis

Proteoglycans (PGs) are composed of a protein moiety and a complex glycosaminoglycan (GAG) polysaccharide moiety. GAG chains are responsible for various biological activities. GAG chains are covalently attached to serine residues of the core protein. The first step in PG biosynthesis is xylosylation of certain serine residues of the core protein. A specific linker tetrasaccharide is then assembled and serves as an acceptor for elongation of GAG chains. If the production of endogenous GAG chains is selectively inhibited, one could determine the role of these endogenous molecules in physiological and developmental functions in a spatiotemporal manner. Biosynthesis of PGs is often blocked with the aid of nonspecific agents such as chlorate, a bleaching agent, and brefeldin A, a fungal metabolite, to elucidate the biological roles of GAG chains. Unfortunately, these agents are highly lethal to model organisms. Xylosides are known to prime GAG chains. Therefore, we hypothesized that modified xylose analogs may able to inhibit the biosynthesis of PGs. To test this, we synthesized a library of novel 4-deoxy-4-fluoroxylosides with various aglycones using click chemistry and examined each for its ability to inhibit heparan sulfate and chondroitin sulfate using Chinese hamster ovary cells as a model cellular system.

A Nanosensor for Ultrasensitive Detection of Oversulfated Chondroitin Sulfate Contaminant in Heparin

Heparin has been extensively used as an anticoagulant for the last eight decades. Recently, the administration of a contaminated batch of heparin caused 149 deaths in several countries including USA, Germany, and Japan. The contaminant responsible for the adverse effects was identified as oversulfated chondroitin sulfate (OSCS). Here, we report a rapid, ultrasensitive method of detecting OSCS in heparin using a nanometal surface energy transfer (NSET) based gold-heparin-dye nanosensor. The sensor is an excellent substrate for heparitinase enzyme, as evidenced by ~70% recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, as the enzyme is inhibited by the contaminant. The newly designed nanosensor can detect as low as 1 × 10(-9) % (w/w) OSCS making it the most sensitive tool to date for the detection of trace amounts of OSCS in pharmaceutical heparins.

A serotonin-induced N-glycan switch regulates platelet aggregation

Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.

4-Deoxy-4-fluoro-xyloside derivatives as inhibitors of glycosaminoglycan biosynthesis

Various 4-deoxy-4-fluoro-xylosides were prepared using click chemistry for evaluating their potential utility as inhibitors of glycosaminoglycan biosynthesis. 2,3-Di-O-benzoyl-4-deoxy-4-fluoro-β-D-xylopyranosylazide, obtained from L-arabinopyranose by six steps, was treated with a wide variety of azide-reactive triple bond-containing hydrophobic agents in the presence of Cu(2+) salt/ascorbic acid, a step known as click chemistry. After click chemistry, benzoylated derivatives were deprotected under Zemplén conditions to obtain 4-deoxy-4-fluoro-xyloside derivatives. A mixture of α:β-isomers of twelve derivatives were then separated on a reverse phase C18 column using HPLC and the resulting twenty four 4-deoxy-4-fluoro-xylosides were evaluated for their ability to inhibit glycosaminoglycan biosynthesis in endothelial cells. We identified two xyloside derivatives that selectively inhibit heparan sulfate and chondroitin sulfate/derman sulfate biosynthesis without affecting cell viability. These novel derivatives can potentially be used to define the biological actions of proteoglycans in model organisms and also as therapeutic agents to combat various human diseases in which glycosaminoglycans participate.