Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity

Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O- and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3'-phosphoadenosine 5'-phosphosulfate as sulfate donor, and cell extract from 6-O-sulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-O-sulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group.     

Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid-degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases-digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.     

Use of sulfated linked cyclitols as heparan sulfate mimetics to probe the heparin/heparan sulfate binding specificity of proteins

Heparin and heparan sulfate (HS) are structurally diverse glycosaminoglycans (GAG) that are known to interact, via unique structural motifs, with a wide range of functionally distinct proteins and modulate their biological activity. To define the GAG motifs that interact with proteins, we assessed the ability of 15 totally synthetic HS mimetics to interact with 10 functionally diverse proteins that bind heparin/HS. The HS mimetics consisted of cyclitol-based pseudo-sugars coupled by linkers of variable chain length, flexibility, orientation, and hydrophobicity, with variations in sulfation also being introduced into some molecules. Three of the proteins tested, namely hepatocyte growth factor, eotaxin, and elastase, failed to interact with any of the sulfated linked cyclitols. In contrast, each of the remaining seven proteins tested exhibited a unique reactivity pattern with the panel of HS mimetics, with tetrameric cyclitols linked by different length alkyl chains being particularly informative. Thus, compounds with short alkyl spacers (2-3 carbon atoms) effectively blocked the interaction of fibroblast growth factor-1 (FGF-1) and lipoprotein lipase with heparin/HS, whereas longer chain spacers (7-10 carbon atoms) were required for optimal inhibition of FGF-2 and vascular endothelial growth factor binding. This effect was most pronounced with the chemokine, interleukin-8, where alkyl-linked tetrameric cyclitols were essentially inactive unless a spacer of >7 carbon atoms was used. The heparin-inhibitable enzymes heparanase and cathepsin G also displayed characteristic inhibition patterns, cathepsin G interacting promiscuously with most of the sulfated cyclitols but heparanase activity being inhibited most effectively by HS mimetics that structurally resemble a sulfated pentasaccharide. These data indicate that a simple panel of HS mimetics can be used to probe the HS binding specificity of proteins, with the position of anionic groups in the HS mimetics being critical.     

Chemoenzymatic synthesis of heparan sulfate and heparin

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays essential physiological and pathophysiological functions. The biosynthesis of HS involves a series of specialised sulfotransferases, an epimerase and glycosyl transferases. The availability of these enzymes offers a promising method to prepare HS polysaccharides and structurally defined oligosaccharides. Given the fact that chemical synthesis of large HS oligosaccharides is extremely difficult, preparation of HS using a chemoenzymatic approach has gained momentum. This review article summarises recent progress on the development of a chemoenzymatic approach to prepare HS and HS oligosaccharides.     

Study of structurally defined oligosaccharide substrates of heparin and heparan monosulfate lyases

The rapid preparation of multimilligram quantities of five heparin-derived oligosaccharides (1–5) is described. These oligosaccharides are the final products obtained from the action of heparin lyase (heparinase, E.C. 4.2.2.7) at its primary sites in the heparin polymer. Five oligosaccharides comprise from 75–85 wt% of commercial porcine mucosal heparins and are recovered in good yield and high purity. Four of these five oligosaccharides were further acted upon at much lower rates by prolonged treatment with heparin lyase or heparan monosulfate lyase (heparitinase, E.C. 4.2.2.8), revealing the subspecificities of these enzymes. These oligosaccharides were used as defined substrates for heparin lyase and heparan monosulfate lyase and their kinetic constants were obtained. Potential applications for these oligosaccharides include their use as defined substrates for purification of heparin monosulfate lyases, and for establishing the catalytic purity of enzyme preparations.     

Conformation and dynamics of heparin and heparan sulfate

The glycosaminoglycans heparin and heparan sulfate contain similar structural units in varying proportions providing considerable diversity in sequence and biological function. Both compounds are alternating copolymers of glucosamine with both iduronate- and glucuronate-containing sequences bearing N-sulfate, N-acetyl, and O-sulfate substitution. Protein recognition of these structurally-diverse compounds depends upon substitution pattern, overall molecular shape, and on internal mobility. In this review particular attention is paid to the dynamic aspects of heparin/heparan sulfate conformation. The iduronate residue possesses an unusually flexible pyranose ring conformation. This extra source of internal mobility creates special problems in rationalization of experimental data for these compounds. We present herein the solution-state NMR parameters, fiber diffraction data, crystallographic data, and molecular modeling methods employed in the investigation of heparin and heparan sulfate. Heparin is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. The literature contains a number of solution and solid-state studies of heparin oligo- and polysaccharides for both isolated heparin species and those bound to protein receptors. These studies indicate a diversity of iduronate ring conformations, but a limited range of glycosidic linkage geometries in the repeating disaccharides. In this sense, heparin exhibits a well-defined overall shape within which iduronate ring forms can freely interconvert. Recent work suggests that computational modeling could potentially identify heparin binding sites on protein surfaces.     

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.     

The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. A structural and biochemical study of the enzyme active site and saccharide substrate specificity

In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157-12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Delta4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.     

Anticoagulant heparan sulfate: structural specificity and biosynthesis

Heparan sulfate (HS) is present on the surface of endothelial and surrounding tissues in large quantities. It plays important roles in regulating numerous functions of the blood vessel wall, including blood coagulation, inflammation response, and cell differentiation. HS is a highly sulfated polysaccharide containing glucosamine and glucuronic/iduronic acid repeating disaccharide units. The unique sulfated saccharide sequences of HS determine its specific functions. Heparin, an analog of HS, is the most commonly used anticoagulant drug. Because of its wide range of biological functions, HS has become an interesting molecule to biochemists, medicinal chemists, and developmental biologists. In this review, we summarize recent progress toward understanding the interaction between HS and blood-coagulating factors, the biosynthesis of anticoagulant HS and the mechanism of action of HS biosynthetic enzymes. Furthermore, knowledge of the biosynthesis of HS facilitates the development of novel enzymatic approaches to synthesize HS from bacterial capsular polysaccharides and to produce polysaccharide end products with high specificity for the biological target. These advancements provide the foundation for the development of polysaccharide-based therapeutic agents.     

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides

Porcine intestinal mucosal heparan sulfate was exhaustivelydepolymerized on a large scale using beparin lyase II (heparinaseII) or heparin lyase III (heparitinase, EC 4.2.2.8 [EC] ). The oligosaccharidemixtures formed with each enzyme were fractionated by low pressuregel permeation chromatography. Size-uniform mixtures of disaccharides,tetrasaccharides, and hexasaccharides were obtained. Each size-fractionatedmixture was then purified on the basis of charge by repetitivesemipreparative strong-anion-exchange high-performance liquidchromatography. This approach has led to the isolation of 13homogenous oligosaccharides. The purity of each oligosaccharidewas demonstrated by the presence of a single peak on analyticalstrong-anion-exchange high-performance liquid chromatographyand reversed polarity capillary electrophoresis. The structuresof these oligosaccharides were established using 500 MHz one-and two-dimensional nuclear magnetic resonance spectroscopy.Three of the thirteen structures that were solved were novelwhile the remaining 10 have been previously described. All ofthe structures obtained using heparin lyase III contained a     

Use of biosynthetic enzymes in heparin and heparan sulfate synthesis

Heparan sulfate and heparin are highly sulfated polysaccharides consisting of repeating disaccharide units of glucuronic acid or iduronic acid that is linked to glucosamine. Heparan sulfate displays a range of biological functions, and heparin is a widely used anticoagulant drug in hospitals. It has been known to organic chemists that the chemical synthesis of heparan sulfate and heparin oligosaccharides is extremely difficult. Recent advances in the study of the biosynthesis of heparan sulfate/heparin offer a chemoenzymatic approach to synthesize heparan sulfate and heparin. Compared to chemical synthesis, the chemoenzymatic method shortens the synthesis and improves the product yields significantly, providing an excellent opportunity to advance the understanding of the structure and function relationships of heparan sulfate. In this review, we attempt to summarize the progress of the chemoenzymatic synthetic method and its application in heparan sulfate and heparin research.     

Interactions between heparan sulfate and proteins: the concept of specificity

Proteoglycan (PG) coreceptors carry heparan sulfate (HS) chains that mediate interactions with growth factors, morphogens, and receptors. Thus, PGs modulate fundamental processes such as cell survival, division, adhesion, migration, and differentiation. This review summarizes recent biochemical and genetic information that sheds new light on the nature of HS-protein binding. Unexpectedly, many interactions appear to depend more on the overall organization of HS domains than on their fine structure.     

Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy

The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.     

Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase

The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).     

LTBP-2 has multiple heparin/heparan sulfate binding sites

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca²⁺ ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca²⁺ dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG–LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.     

Kinetics and specificity of alginate lyases

A purified preparation of the extracellular alginate lyase has been used to study kinetics and specificity towards purified, homopolymeric fragments of alginate. The enzyme preparation from Bacillus circulans 1351 degraded both block types, although with different efficiency, and thus appears to be nonspecific. Addition of calcium ions markedly enhanced the reaction rate for the polymannuronate block but had little or no effect on the reaction with polyguluronate. Michaelis-Menten kinetics are not obeyed in the absence of calcium ions and only for the polymannuronate in the presence of calciumThe study of progress curves in response to variation in substrate and enzyme concentrations strongly suggests that the abalone lyase is subject to a reversible product inhibition.     

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.     

Bivalent and co-operative binding of complement factor H to heparan sulfate and heparin

FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K(d) of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, R(G), of FH in the presence of heparin fragments 18-36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with full-length FH gave K(d) values of 1-3 μM, and similar but weaker K(d) values of 4-20 μM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces.