Jillian R. Brown

Heparin’s anti-inflammatory effects require glucosamine 6- O -sulfation and are mediated by blockade of L- and P-selectins

Heparin has been used clinically as an anticoagulant and antithrombotic agent for over 60 years. Here we show that the potent anti-inflammatory property of heparin results primarily from blockade of P-selectin and L-selectin. Unfractionated heparin and chemically modified analogs were tested as inhibitors of selectin binding to immobilized sialyl Lewis(X) and of cell adhesion to immobilized selectins or thrombin-activated endothelial cells. Compared with unfractionated heparin, the modified heparinoids had inhibitory activity in this general order: over-O-sulfated heparin > heparin > 2-O,3-O-desulfated > or = N-desulfated/N-acetylated heparin > or = carboxyl-reduced heparin > or= N-,2-O,3-O-desulfated heparin >> 6-O-desulfated heparin. The heparinoids also showed similar differences in their ability to inhibit thioglycollate-induced peritonitis and oxazolone-induced delayed-type hypersensitivity. Mice deficient in P- or L-selectins showed impaired inflammation, which could be further reduced by heparin. However, heparin had no additional effect in mice deficient in both P- and L-selectins. We conclude that (a) heparins anti-inflammatory effects are mainly mediated by blocking P- and L-selectin-initiated cell adhesion; (b) the sulfate groups at C6 on the glucosamine residues play a critical role in selectin inhibition; and (c) some non-anticoagulant forms of heparin retain anti-inflammatory activity. Such analogs may prove useful as therapeutically effective inhibitors of inflammation.

Biosynthesis of the Linkage Region of Glycosaminoglycans CLONING AND ACTIVITY OF GALACTOSYLTRANSFERASE II, THE SIXTH MEMBER OF THE β1,3-GALACTOSYLTRANSFERASE FAMILY (β3GalT6)

A family of five β1,3-galactosyltransferases has been characterized that catalyze the formation of Galβ1,3GlcNAcβ and Galβ1,3GalNAcβ linkages present in glycoproteins and glycolipids (β3GalT1, -2, -3, -4, and -5). We now report a new member of the family (β3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found inDrosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, β3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the β3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal β-linked galactose residue. This product, Galβ1,3Galβ is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAβ1,3Galβ1,3Galβ1,4Xylβ-O-Ser), indicating that β3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.

Tumor attenuation by combined heparan sulfate and polyamine depletion

Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemble de novo through a pathway sensitive to the inhibitor, α-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferation in vitro and in vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.

Surfen, a small molecule antagonist of heparan sulfate

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF165-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.

Genistein Improves Neuropathology and Corrects Behaviour in a Mouse Model of Neurodegenerative Metabolic Disease

Background Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein. Methodology/Principal Findings We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed. Conclusions/Significance Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.

Disease-specific non–reducing end carbohydrate biomarkers for mucopolysaccharidoses

A significant need exists for improved biomarkers for differential diagnosis, prognosis and monitoring of therapeutic interventions for mucopolysaccharidoses (MPS), inherited metabolic disorders that involve lysosomal storage of glycosaminoglycans. Here, we report a simple reliable method based on the detection of abundant non-reducing ends of the glycosaminoglycans that accumulate in cells, blood, and urine of MPS patients. In this method, glycosaminoglycans were enzymatically depolymerized releasing unique mono-, di-, or trisaccharides from the non-reducing ends of the chains. The composition of the released mono- and oligosaccharides depends on the nature of the lysosomal enzyme deficiency, and therefore they serve as diagnostic biomarkers. Analysis by liquid chromatography/mass spectrometry allowed qualitative and quantitative assessment of the biomarkers in biological samples. We provide a simple conceptual scheme for diagnosing MPS in uncharacterized samples and a method to monitor efficacy of enzyme replacement therapy or other forms of treatment.

The Caenorhabditis elegans genes sqv-2 and sqv-6, which are required for vulval morphogenesis, encode glycosaminoglycan galactosyltransferase II and xylosyltransferase

In mutants defective in any of eightCaenorhabditis elegans sqv (squashedvulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization ofsqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAβ1,3Galβ1, 3Galβ1,4Xylβ-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans.

Glycan Antagonists and Inhibitors: A Fount for Drug Discovery

ABSTRACT Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.

Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153–164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2stf/f) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1f/f) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2stf/fAlbCre+ mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1f/fAlbCre+ mice. In contrast, Hs6st1f/fAlbCre+ mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.

Expression Patterns of α2,3-Sialyltransferases and α1,3-Fucosyltransferases Determine the Mode of Sialyl Lewis X Inhibition by Disaccharide Decoys

A variety of human adenocarcinomas express sialylated, fucosylated Lewis blood group antigens on cell surface and secreted mucins. Binding of these antigens to P-selectin on platelets is thought to facilitate formation of platelet-tumor emboli in the circulation, which in turn allows sequestration of the tumor cells in the microvasculature. Here we report a pharmacologic approach for blocking these interactions through metabolic inhibition of sialylation. Peracetylated forms of Galβ1,4GlcNAcβ-O-naphthalenemethanol and GlcNAcβ1,3Galβ-O-naphthalenemethanol were taken up by LS180 human colon carcinoma cells, O-deacetylated, and utilized as biosynthetic intermediates, resulting in heterogeneous oligosaccharides. The primed oligosaccharides included sialylated, sulfated, and fucosylated products based on mass spectrometry. Assembly of free oligosaccharides on the glycosides decoyed glycosylation of cellular glycoproteins, as assessed by altered binding of lectins and carbohydrate-specific antibodies. Expression of α2,3-sialylated oligosaccharides on the cell surface was diminished specifically, whereas α2,6-sialylation and fucosylation were not. In U937 lymphoma cells, the glycosides decreased fucosylation without affecting sialylation. The differential inhibitory activities correlated inversely with fucosyltransferase and sialyltransferase activity based on enzyme assays and microarray analysis. Regardless of the mechanism, the disaccharides blocked the cells from forming selectin ligands and inhibited adhesion to immobilized selectins, suggesting that the glycosides might prove useful for interfering with tumor cell adhesion and metastasis.