Chemoenzymatic Design of Heparan Sulfate Oligosaccharides

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C₅-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.     

Heparan sulfate fine structure and specificity of proteoglycan functions

Heparan sulfate chains have markedly heterogeneous structures in which distinct patterns of sulfation determine the binding specificity for ligand proteins. These "fine structures" of heparan sulfate are mainly produced by the regulated introduction of sulfate groups at the N-, 2-O-, 6-O-, and 3-O-positions of the sugar chain. Recent biochemical, histochemical, and genetic studies have demonstrated that different fine structures mediate distinct molecular recognition events to regulate a variety of cellular functions. In this review, we focus on the molecular basis of growth factor control by the sulfation status of heparan sulfate.     

Enzymatic redesigning of biologically active heparan sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.     

Deciphering Mode of Action of Heparanase Using Structurally Defined Oligosaccharides

Heparan sulfate (HS) is a highly sulfated polysaccharide that serves many biological functions, including regulating cell growth and inflammatory responses as well as the blood coagulation process. Heparanase is an enzyme that cleaves HS and is known to display a variety of pathophysiological effects in cancer, diabetes, and Alzheimer disease. The link between heparanase and diseases is a result of its selective cleavage of HS, which releases smaller HS fragments to enhance cell proliferation, migration, and invasion. Despite its importance in pathological diseases, the structural cues in HS that direct heparanase cleavage and the steps of HS depolymerization remain unknown. Here, we sought to probe the substrate specificity of heparanase using a series of structurally defined oligosaccharide substrates. The sites of heparanase cleavage on the oligosaccharide substrates were determined by mass spectrometry and gel permeation chromatography. We discovered that heparanase cleaves the linkage of glucuronic acid linked to glucosamine carrying 6-O-sulfo groups. Furthermore, our findings suggest that heparanase displays different cleavage modes by recognizing the structures of the nonreducing ends of the substrates. Our results deepen the understanding of the action mode of heparanase.     

The dominating role of N-deacetylase/N-sulfotransferase 1 in forming domain structures in heparan sulfate

Heparan sulfate (HS) is a highly sulfated polysaccharide participated in essential physiological functions from regulating cell growth to blood coagulation. HS contains sulfated domains known as N-S domains and low sulfate domains known as N-Ac domains. The distribution of the domain structures is likely governed by the action of glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here, we sought to determine the substrate specificity of NDST using model substrates and recombinant NDST protein. We discovered that NDST-1 carries out the modification in a highly ordered fashion. The enzyme sulfates the substrate from the nonreducing end toward the reducing end consecutively, leading to the product with a cluster of N-sulfo glucosamine residues. Furthermore, a preexisting N-sulfo glucosamine residue prevents the action of NDST-1 at the residues immediately located at the nonreducing end, allowing the formation of an N-Ac domain. Our results provide the long sought evidence for understanding the formation of sulfated versus nonsulfated domains in the HS isolated from cells and tissues. The study demonstrates the regulating role of NDST-1 in mapping the sulfation patterns of HS.     

Human heparanase. Purification, characterization, cloning, and expression.

Heparan sulfate and heparan sulfate proteoglycans are present in the extracellular matrix as well as on the external cell surface. They bind various molecules such as growth factors and cytokines and modulate the biological functions of binding proteins. Heparan sulfate proteoglycans are also important structural components of the basement membrane. Heparanase is an endo-beta-D-glucuronidase capable of cleaving heparan sulfate and has been implicated in inflammation and tumor angiogenesis and metastasis. In this study, we report the purification of a human heparanase from an SV40-transformed embryonic fibroblast cell line WI38/VA13 by four sequential column chromatographies. The activity was measured by high speed gel permeation chromatography of the degradation products of fluorescein isothiocyanate-labeled heparan sulfate. The enzyme was purified to homogeneity, yielding a peptide with an apparent molecular mass of 50 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Using the amino acid sequences of the N-terminal and internal heparanase peptides, a cDNA coding for human heparanase was cloned. NIH3T3 and COS-7 cells stably transfected with pBK-CMV expression vectors containing the heparanase cDNA showed high heparanase activities. The homology search revealed that no homologous protein had been reported.     

Characterization of heparanase from a rat parathyroid cell line

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-beta-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1-2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.     

Implications of heparan sulfate and heparanase in neuroinflammation

Heparan sulfate proteoglycans (HSPGs), expressed on the cell surface and in the extracellular matrix of most animal tissues, have essential functions in development and homeostasis, and have been implicated in several pathological conditions. The functions of HSPGs are mainly mediated through interactions of the heparan sulfate (HS) polysaccharide side chains with different protein ligands. The molecular structure of HS is highly diverse, expressed in a cell-type specific manner. The flexible yet controlled structure of HS is primarily generated through a strictly regulated biosynthesis process and is further modified post-synthetically, such as desulfation by endosulfatases and fragmentation by heparanase. Heparanase is an endo-glucuronidase expressed in all tissues. The enzyme has been found up-regulated in a number of pathological conditions, implying a role in diseases mainly through degradation of HS. Emerging evidence demonstrates important roles of HS and heparanase in inflammatory reactions, particularly in the regulation of leukocyte activation and extravasation. Neuroinflammation is a common feature of various central nervous system disorders, thus it is a great interest to understand the implications of HS and heparanase in neuroinflammation.     

Sulfation pattern in glycosaminoglycan: Does it have a code?

Heparan sulfate chains (HS) are initially synthesized on core proteins as linear polysaccharides composed of glucuronic acid--N-acetylglucosamine repeating units and subjected to marked structural modification by sulfation (N-, 2-O-, 6-O-, 3-O-sulfotransferases) and epimerization (C5-epimerase) at the Golgi lumen and further by desulfation (6-O- endosulfatase) at the cell surface, after which divergent fine structures are generated. The expression patterns and specificity of the modifying enzymes are, at least partly, responsible for the elaboration of these fine structures of heparan sulfate. HS interacts with many proteins including growth factors (GF) and morphogens through specific fine structures. Recent biochemical and genetic studies have presented evidence that HS plays important roles in cell behavior and organogenesis. In knock-down experiments of heparan sulfate 6-O-sulfotransferase, 6-O-sulfated units in HS have been shown to act as a stimulator or suppressor according to individual GF/morphogen signaling systems.     

Enzymatic placement of 6-O-sulfo groups in heparan sulfate

Heparan sulfate is a highly sulfated polysaccharide that exhibits important physiological and pathological functions. The glucosamine residue of heparan sulfate can carry sulfo groups at the 2-N, 3-O, and 6-O positions, leading to diverse polysaccharide structures. 6-O-Sulfation at the glucosamine residue contributes to a wide range of biological functions. Here, we report a method for controlling the positioning of 6-O-sulfo groups in oligosaccharides. This was achieved by synthesizing oligosaccharide backbones from a disaccharide building block utilizing glycosyltransferases followed by modifications using heparan sulfate N-sulfotransferase and 6-O-sulfotransferases. This method offers a viable approach for preparing heparan sulfate oligosaccharides with precisely located 6-O-sulfo groups.     

Organ-specific sulfation patterns of heparan sulfate generated by extracellular sulfatases Sulf1 and Sulf2 in mice

Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here, we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knock-out mice. Significant increases in ΔUA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1(-/-) mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2(-/-) mice. In addition, increases in ΔUA-GlcNS6S were seen in the Sulf1(-/-) lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in eight organs of wild-type mice were highly correlated with increases in ΔUA2S-GlcNS6S in the corresponding organs of knock-out mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1(-/-) mice than in Sulf2(-/-) mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently, the sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knock-out mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.     

Regulation of urokinase/urokinase receptor interaction by heparin-like glycosaminoglycans

We show here that the interaction between the urokinase-type plasminogen activator and its receptor, which plays a critical role in cell invasion, is regulated by heparan sulfate present on the cell surface and in the extracellular matrix. Heparan sulfate oligomers showing a composition close to the dimeric repeats of heparin (glucosamine-NSO(3)(6-OSO(3))-iduronic acid(2-OSO(3))) n = 5 and n > 5, where iduronic acid may alternate with glucuronic acid, exhibit affinity for urokinase plasminogen activator and confer specificity on urokinase/urokinase receptor interaction. Cell surface clearance of heparan sulfate reduces the affinity of such interaction with a parallel decrease of specific urokinase binding in the presence of an unaltered expression of receptor. Transfection of human urokinase plasminogen activator receptor in normal Chinese hamster ovary fibroblasts and in Chinese hamster ovary cells defective for the synthesis of sulfated glycosaminoglycans results in specific urokinase/receptor interaction only in nondefective cells. Heparan sulfate/urokinase and receptor/urokinase interactions exhibit similar K(d) values. We concluded that heparan sulfate functions as an adaptor molecule that confers specificity on urokinase/receptor binding.     

Regulation of platelet heparanase during inflammation: Role of pH and proteinases

Heparan sulfate is rapidly degraded by an endoglycosidase (heparanase) secreted by activated platelets. Since the cleavage and release of heparan sulfate would profoundly alter the local physiology of the endothelium, platelet heparanase activity should be tightly regulated. Consistent with this hypothesis, platelet heparanase was found to degrade endothelial cell heparan sulfate at pH 6.0 but not at pH 7.4, even though 25% of maximum activity was detected at pH 7.4. Loss of heparanase activity occurred rapidly (t_1/2 ≅ 20 min) and reversibly at physiologic pH but did not occur at acidic pH (<7.0). Inactivation of heparanase at pH 7.4 did not affect heparin binding and was reversed by 0.5 M NaCl or by heparan sulfate but not by chondroitin sulfate, suggesting inactive heparanase could be tethered on cell surfaces and the function regulated by heparan sulfate. Heparanase was gradually inactivated by trypsin and urokinase (t_1/2 = 5 h) but resisted cleavage by leukocyte cathepsin G, leukocyte elastase, plasmin, and thrombin. These findings are consistent with a model in which platelet heparanase is active at the low pH of inflammation but inactive under physiologic conditions preventing inadvertent cleavage of heparan sulfate and loss of physiologic functions of endothelial cells. J. Cell. Physiol. 175:255–267, 1998. © 1998 Wiley-Liss, Inc.     

Heparanase expression in invasive trophoblasts and acute vascular damage

Heparan sulfate proteoglycans play a pivotal role in tissue function, development, inflammation, and immunity. We have identified a novel cDNA encoding human heparanase, an enzyme thought to cleave heparan sulfate in physiology and disease, and have located the HEP gene on human chromosome 4q21. Monoclonal antibodies against human heparanase located the enzyme along invasive extravillous trophoblasts of human placenta and along endothelial cells in organ xenografts targeted by hyperacute rejection, both sites of heparan sulfate digestion. Heparanase deposition was evident in arterial walls in normal tissues; however, vascular heparan sulfate cleavage was coincident with heparanase enzyme during inflammatory episodes. These findings suggest that heparanase elaboration and control of catalytic activity may contribute to the development and pathogenesis of vascular disease and suggest that heparanase intervention might be a useful therapeutic target.     

Defective heparan sulfate biosynthesis and neonatal lethality in mice lacking N-deacetylase/N-sulfotransferase-1

Heparan sulfate is a sulfated polysaccharide present on most cell surfaces and in the extracellular matrix. In vivo functions of heparan sulfate can be studied in mouse strains lacking enzymes involved in the biosynthesis of heparan sulfate. Glucosaminyl N-deacetylase/N-sulfotransferase (NDST) catalyzes the first modifying step in the biosynthesis of the polysaccharide. This bifunctional enzyme occurs in several isoforms. We here report that targeted gene disruption of NDST-1 in the mouse results in a structural alteration of heparan sulfate in most basement membranes as revealed by immunohistochemical staining of fetal tissue sections using antibodies raised against heparan sulfate. Biochemical analysis of heparan sulfate purified from fibroblast cultures, lung, and liver of NDST-1-deficient embryos demonstrated a dramatic reduction in N-sulfate content. Most NDST-1-deficient embryos survive until birth; however, they turn out to be cyanotic and die neonatally in a condition resembling respiratory distress syndrome. In addition, a minor proportion of NDST-1-deficient embryos die during the embryonic period. The cause of the embryonic lethality is still obscure, but incompletely penetrant defects of the skull and the eyes have been observed.     

Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.     

Poly(ethylene glycol acrylate)‐functionalized hydrogels for heparan sulfate oligosaccharide recognition

Heparan sulfates are complex polysaccharides belonging to the family of glycosaminoglycans that participate to the regulation of cell behavior and tissue homeostasis. The biological activities conferred to heparan sulfates are largely dependent on the content and positioning of the sulfate groups along their saccharidic units. At present, identification of particular sulfation patterns in biologically relevant heparan sulfate sequences remains challenging. Although several approaches for structure analysis exist, the complexity of heparan sulfates makes new and original approaches still required. Here, we used molecular imprinting technologies to prepare a library of polyethylene glycol acrylate functionalized hydrogels with the aim to investigate their applicability as specific recognizing systems for fondaparinux, a synthetic pentasaccharide analog to the antithrombin binding site of heparin. Adequate choice of the hydrogel composition and controlling rebinding conditions were important determinants for improving the sulfated oligosaccharide recognition specificity and selectivity. Our results suggest that molecular imprinting approaches could be a possibility for the specific recognition of biologically active sequences in heparan sulfates.     

乙酰肝素酶的生物学特性的研究进展

乙酰肝素酶是切割哺乳动物细胞中硫酸肝素蛋白多糖侧链——硫酸乙酰肝素的内源性糖苷酶,是抗肿瘤转移的理想靶点。本就乙酰肝素酶的分子结构特点、亚细胞定位、活性调控机制、与肿瘤转移的关系、底物特异性和抑制剂开发等方面的研究进展进行了综述。