Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.     

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.     

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.     

Kinetic and structural studies on interactions between heparin or heparan sulfate and proteins of the hedgehog signaling pathway

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and regulation of the tissue distribution of signaling molecules. In this study, we use surface plasmon resonance (SPR) to study interactions of HS and structurally related heparin with proteins in the Hedgehog signaling pathway. SPR analysis shows that heparin binds with different affinities to active fragments of the proteins Hedgehog (Hh), Interference Hedgehog (Ihog), Cam-related/Down-regulated by Oncogenes (CDO), and Sonic Hedgehog (Shh). Solution competition studies show that the minimum size of a heparin oligosaccharide capable of interacting with Ihog is larger than a tetrasaccharide and for interacting with Shh is larger than an octasaccharide. In comparison with heparin, Ihog and Shh exhibited a lower affinity for HS than for heparin, and CDO and Hh exhibit negligible binding to HS. This study clearly demonstrates Shh and Ihog are heparin and HS binding proteins and that both molecules preferentially bind heparin or HS having a high level of sulfation.     

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.     

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.     

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.     

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     

Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

Structural requirements for heparin/heparan sulfate binding to type V collagen

Collagen-proteoglycan interactions participate in the regulation of matrix assembly and in cell-matrix interactions. We reported previously that a fragment (Ile824-Pro950) of the collagen alpha1(V) chain, HepV, binds to heparin via a cluster of three major basic residues, Arg912, Arg918, and Arg921, and two additional residues, Lys905 and Arg909 (Delacoux, F., Fichard, A., Cogne, S., Garrone, R., and Ruggiero, F. (2000) J. Biol. Chem. 275, 29377-29382). Here, we further characterized the binding of HepV and collagen V to heparin and heparan sulfate by surface plasmon resonance assays. HepV bound to heparin and heparan sulfate with a similar affinity (KD approximately 18 and 36 nM, respectively) in a cation-dependent manner, and 2-O-sulfation of heparin was shown to be crucial for the binding. An octasaccharide of heparin and a decasaccharide of heparan sulfate were required for HepV binding. Studies with HepV mutants showed that the same basic residues were involved in the binding to heparin, to heparan sulfate, and to the cell surface. The contribution of Lys905 and Arg909 was found to be significant. The triple-helical peptide GPC(GPP)5G904-R918(GPP)5GPC-NH2 and native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.     

Structural determinants of heparan sulfate interactions with Slit proteins

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican-Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin-Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin-heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (approximately 60% at 2 microM concentration) was attained with a dodecasaccharide.     

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.     

Preparation of heparin/heparan sulfate oligosaccharides with internal N-unsubstituted glucosamine residues for functional studies

The rare N-unsubstituted glucosamine (GlcNH (3)(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. However, it is difficult to prepare naturally-occuring, GlcNH(3)(+)-containing oligosaccharides from HS because of their low abundance, as well as the inherent problems in both excising and identifying them. Therefore, the ability to chemically generate a series of structurally-defined oligosaccharides containing GlcNH(3)(+) residues would greatly contribute to investigating their natural role in HS. In this study, a series of heparin/HS oligosaccharides, from dp6 up to dp16 in length that possess internal GlcNH(3)(+) residues were prepared by a combination of chemical modification and heparinase I digestion. Purification and structural analysis of the major species derived from the octa- to dodeca-saccharide size fractions indicated the introduction of between 1 and 3 internal GlcNH(3)(+) residues per oligosaccharide. In addition, a GlcNH(3)(+) residue was selectively introduced into an internal position in a tetrasaccharide species by direct chemical modification. This selectivity has potential as an alternative procedure for preparing internally-modified oligosaccharides of various lengths. The utility of such oligosaccharides was demonstrated by a comparison of the binding of three different tetrasaccharide species containing 0, 1 and 2 free amino groups to the NK1 truncated variant of hepatocyte growth factor/scatter factor.     

Novel heparin/heparan sulfate mimics as inhibitors of HGF/SF-induced MET activation

The synthesis of simple, non-sugar glycosaminoglycan (GAG) mimics has been achieved and the analogues evaluated for their ability to inhibit the activation of the MET receptor by hepatocyte growth factor/scatter factor (HGF/SF).     

Synthetic heparin and heparan sulfate oligosaccharides and their protein interactions

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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.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.