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.     

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.     

Paper disk assay for glycosaminoglycan sulfotransferases

A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the [35S]sulfate-labeled products are precipitated and then separated from a sulfate donor ([35S]PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.     

Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3?nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities.     

Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis

Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate are polysaccharide chains that are attached to core proteins to form proteoglycans. The biosynthesis of GAGs is a multistep process that includes the attachment of sulfate groups to specific positions of the polysaccharide chains by sulfotransferases. Heparan-sulfate and heparan sulfate-sulfotransferases play important roles in growth factor signaling and animal development. However, the biological importance of chondroitin sulfation during mammalian development and growth factor signaling is poorly understood. We show that a gene trap mutation in the BMP-induced chondroitin-4-sulfotransferase 1 (C4st1) gene (also called carbohydrate sulfotransferase 11 - Chst11), which encodes an enzyme specific for the transfer of sulfate groups to the 4-O-position in chondroitin, causes severe chondrodysplasia characterized by a disorganized cartilage growth plate as well as specific alterations in the orientation of chondrocyte columns. This phenotype is associated with a chondroitin sulfation imbalance, mislocalization of chondroitin sulfate in the growth plate and an imbalance of apoptotic signals. Analysis of several growth factor signaling pathways that are important in cartilage growth plate development showed that the C4st1(gt/gt) mutation led to strong upregulation of TGFbeta signaling with concomitant downregulation of BMP signaling, while Indian hedgehog (Ihh) signaling was unaffected. These results show that chondroitin 4-O-sulfation by C4st1 is required for proper chondroitin sulfate localization, modulation of distinct signaling pathways and cartilage growth plate morphogenesis. Our study demonstrates an important biological role of differential chondroitin sulfation in mammalian development.     

Heparan sulfate 3-O-sulfation: A rare modification in search of a function

Many protein ligands bind to heparan sulfate, which results in their presentation, protection, oligomerization or conformational activation. Binding depends on the pattern of sulfation and arrangement of uronic acid epimers along the chains. Sulfation at the C3 position of glucosamine is a relatively rare, yet biologically significant modification, initially described as a key determinant for binding and activation of antithrombin and later for infection by type I herpes simplex virus. In mammals, a family of seven heparan sulfate 3-O-sulfotransferases installs sulfate groups at this position and constitutes the largest group of sulfotransferases involved in heparan sulfate formation. However, to date very few proteins or biological systems have been described that are influenced by 3-O-sulfation. This review describes our current understanding of the prevalence and structure of 3-O-sulfation sites, expression and substrate specificity of the 3-O-sulfotransferase family and the emerging roles of 3-O-sulfation in biology.     

Multi-faceted substrate specificity of heparanase

Heparan sulfate is a highly sulfated polysaccharide abundantly present in the extracellular matrix. Heparan sulfate consists of a disaccharide repeating unit of glucosamine and glucuronic and iduronic acid residues. The functions of heparan sulfate are largely dictated by its size as well as the sulfation patterns. Heparanase is an enzyme that cleaves heparan sulfate polysaccharide into smaller fragments, regulating the functions of heparan sulfate. Understanding the substrate specificity plays a critical role in dissecting the biological functions of heparanase and heparan sulfate. The prevailing view is that heparanase recognizes specific sulfation patterns in heparan sulfate. However, emerging evidence suggests that heparanase is capable of varying its substrate specificities depending on the saccharide structures around the cleavage site. The plastic substrate specificity suggests a complex role of heparanase in regulating the structures of heparan sulfate in matrix biology.     

Sulfation of environmental estrogen-like chemicals by human cytosolic sulfotransferases

To investigate whether sulfation, a major Phase II detoxification pathway in vivo, can be employed as a means for the inactivation/disposal of environmental estrogens, recombinant human cytosolic sulfotransferases were prepared and tested for enzymatic activities with bisphenol A, diethylstilbestrol, 4-octylphenol, p-nonylphenol, and 17alpha-ethynylestradiol as substrates. Of the seven recombinant enzymes examined, only SULT1C sulfotransferase #1 showed no activities toward the environmental estrogens tested. Among the other six sulfotransferases, the simple phenol (P)-form phenol sulfotransferase and estrogen sulfotransferase appeared to be considerably more active toward environmental estrogens than the other four sulfotransferases. Metabolic labeling experiments revealed the sulfation of environmental estrogens and the release of their sulfated derivatives by HepG2 human hepatoma cells. Moreover, sulfated environmental estrogens appeared to be incapable of penetrating through the HepG2 cell membrane.     

Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. Implications for heparan sulfate proteoglycan biosynthesis

We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.     

Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1

Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO(3)) from 3'-phosphoadenosine 5'-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate. This structure reveals residues critical for 3'-phosphoadenosine 5'-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.     

Crystal structure-based studies of cytosolic sulfotransferase

Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities. Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans. A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates. Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1. These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity. These crystal structures introduce a new era of the study of the sulfotransferases.     

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.     

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.     

A peptide found by phage display discriminates a specific structure of a trisaccharide in heparin

A number of recent studies have shown that heparan sulfate can control several important biological events on the cell surface through changes in sulfation pattern. The in vivo modification of sugar chains with sulfates, however, is complicated, and the discrimination of different sulfation patterns is difficult. Heparin, which is primarily produced by mast cells, is closely approximated by the structural analog heparan sulfate. Screening of heparin-associating peptides using phage display and antithrombin-bound affinity chromatography identified a peptide, heparin-associating peptide Y (HappY), that acts as a target of immobilized heparin. The peptide consists of 12 amino acid residues with characteristic three arginines and exclusively binds to heparin and heparan sulfate but does not associate with other glycosaminoglycans. HappY recognizes three consecutive monosaccharide residues in heparin through its three arginine residues. HappY should be a useful probe to detect heparin and heparan sulfate in studies of glycobiology.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

Glycosaminoglycan sulfotransferases in human and animal sera

Heparan sulfate, keratan sulfate, chondroitin, chondroitin 4/6-sulfate (80% 4-sulfate and 20% 6-sulfate), and UDP-N-acetylgalactosamine 4-sulfate were used as acceptors for the measurement of 3'-phosphoadenylyl sulfate: glycosaminoglycan sulfotransferase activities in human serum. Chromatographic fractionation of the serum followed by determination of the sulfotransferase activities demonstrated the existence of at least four different sulfotransferases capable of introducing sulfate to 1) position 6 of the internal N-acetylgalactosamine units of chondroitin, 2) position 6 of the nonreducing terminal N-acetylgalactosamine 4-sulfate unit of chondroitin 4/6-sulfate, 3) position 2 (amino group) of the glucosamine units in heparan sulfate, and 4) the sugar units in keratan sulfate, respectively. The fourth activity was separated into two subfractions with different specificities for the structure of neighboring sugars of the sulfate-accepting sugar units. No major variations in the sulfotransferase activities on added receptors were found to occur in sera from individuals 22-41 years old. In contrast, the activities in sera of various mammalian and avian species showed a species-specific variation. With mouse skin fibroblasts cultured in serum-free medium, preferential secretion of several sulfotransferases could be demonstrated. The results, taken together, suggest that the appearance of the sulfotransferases in serum is not a fortuitous event due to nonspecific cell death, but the result of an elaborate mechanism for enzyme secretion by a cell or tissue system.     

Continuous fluorometric assay of phenol sulfotransferase.

Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.     

Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A sulfates N-unsubstituted glucosamine residues

3-O-Sulfation of glucosamine by heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1) is the key modification in anticoagulant heparan sulfate synthesis. However, the heparan sulfates modified by 3-OST-2 and 3-OST-3A, isoforms of 3-OST-1, do not have anticoagulant activity, although these isoforms transfer sulfate to the 3-OH position of glucosamine residues. In this study, we characterize the substrate specificity of purified 3-OST-3A at the tetrasaccharide level. The 3-OST-3A enzyme was purified from Sf9 cells infected with recombinant baculovirus containing 3-OST-3A cDNA. Two 3-OST-3A-modified tetrasaccharides were purified from the 3-O-(35)S-sulfated heparan sulfate that was digested by heparin lyases. These tetrasaccharides were analyzed using nitrous acid and enzymatic degradation combined with matrix-assisted laser desorption/ionization-mass spectrometry. Two novel tetrasaccharides were discovered with proposed structures of DeltaUA2S-GlcNS-IdoUA2S-[(35)S]GlcNH(2)3S and DeltaUA2S-GlcNS-IdoUA2S-[3-(35)S]GlcNH(2)3S6S . The results demonstrate that 3-OST-3A sulfates N-unsubstituted glucosamine residues, and the 3-OST-3A modification sites are probably located in defined oligosaccharide sequences. Our study suggests that oligosaccharides with N-unsubstituted glucosamine are precursors for sulfation by 3-OST-3A. The intriguing linkage between N-unsubstituted glucosamine and the 3-O-sulfation by 3-OST-3A may provide a clue to the potential biological functions of 3-OST-3A-modified heparan sulfate.     

Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST) gene. Structure, expression, and function in the formation of the tracheal system

Heparan sulfate, one of the most abundant components of the cell surface and the extracellular matrix, is involved in a variety of biological processes such as growth factor signaling, cell adhesion, and enzymatic catalysis. The heparan sulfate chains have markedly heterogeneous structures in which distinct sequences of sulfate groups determine specific binding properties. Sulfation at each different position of heparan sulfate is catalyzed by distinct enzymes, sulfotransferases. In this study, we identified and characterized Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST). The deduced primary structure of dHS6ST exhibited several common features found in those of mammalian HS6STs. We confirmed that, when the protein encoded by the cDNA was expressed in COS-7 cells, it showed HS6ST activity. Whole mount in situ hybridization revealed highly specific expression of dHS6ST mRNA in embryonic tracheal cells. The spatial and temporal pattern of dHS6ST expression in these cells clearly resembles that of the Drosophila fibroblast growth factor (FGF) receptor, breathless (btl). RNA interference experiments demonstrated that reduced dHS6ST activity caused embryonic lethality and disruption of the primary branching of the tracheal system. These phenotypes were reminiscent of the defects observed in mutants of FGF signaling components. We also show that FGF-dependent mitogen-activated protein kinase activation is significantly reduced in dHS6ST double-stranded RNA-injected embryos. These findings indicate that dHS6ST is required for tracheal development in Drosophila and suggest the evolutionally conserved roles of 6-O-sulfated heparan sulfate in FGF signaling.