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R J Linhardt

Publications and source records attributed to R J Linhardt.

At least 37 records · Page 2Linked to original sources
PubMed1997

Interaction of fibroblast growth factor-1 and related peptides with heparan sulfate and its oligosaccharides.

Fibroblast growth factors (FGFs) are a family of angiogenic and mitogenic proteins that promote cell division. The binding of FGFs to the heparan sulfate of cell-surface-bound proteoglycans appears to be critical for their activity. The interaction of fibroblast growth factor-1 (FGF-1 or aFGF) using heparin lyase-derived oligosaccharides from heparan sulfate was investigated. FGF-1 was also shown to protect sequences in heparan sulfate from heparin lyase digestion and protected oligosaccharide products of octasaccharide and decasaccharide size were recovered by FGF-1 affinity chromatography, suggesting that the high-affinity binding of heparan sulfate to FGF-1 resides within an octasaccharide sequence. The FGF-1 binding affinity of heparan sulfate is reduced compared to heparin presumably due to the absence of 6-sulfate groups in heparan sulfate. Inspection of the FGF-1 heparan sulfate binding domain shows that the majority of interacting amino acids are contained within a 20-amino-acid sequence that folds back upon itself (because of three turns) forming a triangular shaped cup of positive charge. The importance of FGF-1 binding site topology was investigated using three synthetic peptide mimics of the FGF-1 glycosaminoglycan (GAG) binding site. Heparan sulfate affinity chromatography and isothermal titration calorimetry, used to measure binding thermodynamics, demonstrated that a synthetic peptide analogous to the GAG binding site in FGF-1 bound tightly to heparan sulfate. A peptide containing a D-proline in place of L-proline bound with considerably reduced affinity, presumably due to the altered structure of the second turn in the binding site. A cyclic peptide, expected to be topologically most similar to the triangular GAG binding site in FGF-1, bound with the highest affinity to heparan sulfate. These data suggest the triangular topology of the GAG binding site in FGF is critical for its interaction with heparan sulfate. Analysis of known GAG binding sites in 25 proteins using the Chou-Fasman algorithm show that these sites commonly contain turns.

Amino Acid Sequence↗
PubMed1997

Chemical microdetermination of heparin in plasma.

A new, simple, and highly sensitive method for the determination of heparin has been established. Heparin was first converted into unsaturated disaccharides through the action of heparin lyases I, II, and III. A major trisulfated unsaturated disaccharide product results, consistent with structural analysis of a number of pharmaceutical heparins using one- and two-dimensional 1H NMR spectroscopy. This disaccharide was analyzed by HPLC using fluorometric postcolumn derivatization. The correlation between the amount of this trisulfated unsaturated disaccharide and anticoagulant activity of heparin as measured by anti-IIa was determined. The analysis of these pharmaceutical heparins showed a linear correlation between both HPLC and bioassay. This HPLC method was then applied to a pharmacokinetic study of heparin intravenously administered to rabbits.

Animals↗
PubMed1997

Pattern and spacing of basic amino acids in heparin binding sites.

Glycosaminoglycan (GAG)-protein interactions regulate a myriad of physiologic and pathologic processes, yet an understanding of how these molecules interact is lacking. The role of the pattern and spacing of basic amino acids (arginine (R) and lysine (K)) in heparin binding sites was investigated using peptide analogs as well as by examining known heparin binding sites. Peptides having the general structure R(n)W (n = 3-9, where tyrosine (W) was added for peptide detection) were synthesized and their interaction with heparin was determined by isothermal titration calorimetry. Binding affinity increased with increasing number of R residues. A 9-mer of R (R9W) bound as tightly to heparin as acidic fibroblast growth factor under physiologic conditions. Despite their high affinity for heparin, long stretches of basic amino acids are uncommon in heparin binding proteins. Known heparin binding sites most commonly contain single isolated basic amino acids separated by one nonbasic amino acid. Peptides having the structure, H3CCONH-GRRG(m)RRG(5-m)-CONH2 (denoted as the RRG(m)RR peptide series) and H3CCONH-GRRRG(m)RG(5-m)-CONH2 (denoted as the RRRG(m)R peptide series), where m = 0-5, were synthesized to test the hypothesis that the spacing of basic amino acids in heparin binding sites is optimally arranged to interact with different GAGs. The peptides, in both the -RRG(m)RR- and -RRRG(m)R- peptide series, when m = 0, bound most tightly with heparin, as measured by affinity chromatography. In contrast, the -RRG(m)RR-peptide series interacted most tightly with heparan sulfate when m = 0 or 1, whereas the -RRRG(m)R- peptide series bound tightest when m = 3. These results are consistent with our understanding of heparin and heparan sulfate structure. A highly sulfated GAG, such as heparin, interacts most tightly with peptides (or peptide sequences within proteins) containing a complementary binding site of high positive charge density. Heparan sulfate, having fewer and more highly spaced negatively charged groups, interacts most tightly with a complementary site on a peptide (or peptide sequences with proteins) that has more widely spaced cationic residues.

Amino Acid Sequence↗
PubMed1997

Glycosaminoglycans can influence fibroblast growth factor-2 mitogenicity without significant growth factor binding.

Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity.

Animals↗
PubMed1997

Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species.

This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain --> 4)-beta-D-GlcAp-(1 --> 4)-alpha-D-GlcNp-(1 --> 4)-beta-D-GlcAp-(1 --> low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues.

Amino Sugars↗
PubMed1997

Exploration of the action pattern of Streptomyces hyaluronate lyase using high-resolution capillary electrophoresis.

Hyaluronic acid was treated exhaustively with a hyaluronate lyase (hyaluronidase, EC 4.2.2.1) from Streptomyces hyalurolyticus to obtain a tetrasaccharide and a hexasaccharide product in a molar ratio of 1 to 1.2. The tetrasaccharide product was fluorescently labeled at the reducing end by reductive amination with 7-amino 1,3-naphthalene disulfonic acid (AGA) and the structure of the conjugate was determined spectroscopically. Partial treatments of hyaluronic acid with hyaluronate lyase afforded complex mixtures of oligosaccharides that were similarly fluorescently labeled. These labeled oligosaccharide mixtures were analyzed using high-resolution capillary electrophoresis. The resulting electropherograms showed the content of each hyaluronic acid derived oligosaccharide, having a degree of polymerization (dp) from 4 to 50, throughout the enzymatic reaction. Computer simulation studies gave comparable kinetic profiles suggesting that hyaluronate lyase exhibits a random endolytic action pattern. Interestingly, oligosaccharides of certain size (dp) were under-represented in these oligosaccharide mixtures suggesting that linkages at spacings of 10 to 12 saccharide units are somewhat resistant to this enzyme. The cause of this resistance might be the result of secondary or higher order structural features present in the hyaluronic acid polymer.

Carbohydrate Sequence↗
PubMed1997

Polysulfated carbohydrates analyzed as ion-paired complexes with basic peptides and proteins using electrospray negative ionization mass spectrometry.

Electrospray ionization mass spectrometry was used in the negative ion mode to analyze complexes of sucrose octasulfate, sucrose heptasulfate and sulfated alpha-, beta- and gamma-cyclodextrins with synthetically prepared basic peptides, the basic protein ubiquitin and polyamines. The spectra presented demonstrate that complexes with these basic molecules facilitate the analysis of these polysulfated oligosaccharides. Stable (1:1) complexes result from the ion pairing between the protonated basic arginine and lysine residues of the peptide and the anionic sulfate groups of the polysulfated oligosaccharides. Fragmentation of the polysulfated oligosaccharides resulting in the loss of SO3 could be suppressed by controlling the experimental conditions, such as the nozzle-skimmer voltage, used to obtain the spectra. In the absence of fragmentation, it was possible to obtain data on the purity of sucrose octasulfate and sucrose heptasulfate as well as the distribution of the sulfated cyclodextrins. The confounding presence of sodium counter-ions is also eliminated using this method. Complete chemical sulfation of oligosaccharides is difficult to achieve. Thus, data on sample purity are essential for the characterization of sulfated oligosaccharides used as pharmaceutical agents.

Carbohydrates↗
PubMed1997

Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate.

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.

Amino Acid Sequence↗
PubMed1997

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides.

Porcine intestinal mucosal heparan sulfate was exhaustively depolymerized on a large scale using heparin lyase II (heparinase II) or heparin lyase III (heparitinase, EC 4.2.2.8). The oligosaccharide mixtures formed with each enzyme were fractionated by low pressure gel permeation chromatography. Size-uniform mixtures of disaccharides, tetrasaccharides, and hexasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semipreparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 13 homogenous oligosaccharides. The purity of each oligosaccharide was demonstrated by the presence of a single peak on analytical strong-anion-exchange high-performance liquid chromatography and reversed polarity capillary electrophoresis. The structures of these oligosaccharides were established using 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy. Three of the thirteen structures that were solved were novel while the remaining 10 have been previously described. All of the structures obtained using heparin lyase III contained a delta UAp residue (where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid) at their nonreducing termini. Structures obtained using heparin lyase II contained both delta UAp and delta UAp2S (where S is sulfate) at their nonreducing termini. These results are consistent with the reported specificity of both enzymes.

Animals↗
PubMed1997

Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor.

Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

Animals↗
PubMed1997

Heparin-dependent fibroblast growth factor activities: effects of defined heparin oligosaccharides.

Heparin and related molecules have been identified as important participants in fibroblast growth factor (FGF) signaling although the mechanisms of action remain unclear. We have used heparin oligosaccharides to examine steps in the signaling process which could be affected by the polysaccharide. Immobilized FGF-1 and FGF-2 bound all sizes of oligosaccharides tested, ranging from tetrasaccharide to decasaccharide, at physiological salt concentration. Each group of oligosaccharide was eluted from the FGF affinity columns in several peaks, and larger oligosaccharides showed higher apparent affinity for the immobilized growth factors compared to the shorter ones. Heparin hexasaccharides were the smallest fragments providing complete protection of FGF-1 and FGF-2 against trypsin digestion. Tetrasaccharides, however, were able to provide partial protection. The requirement of heparin for ligand-receptor interaction was evaluated in receptor binding assays using Sf9 insect cells engineered to overexpress different recombinant FGF receptor (FGFR) species including FGFR1 beta, FGFR1 alpha or FGFR4 at the cell surface. In these assays hexasaccharides were the smallest fragments capable of stimulating FGF-receptor interaction. Over the range of concentrations examined, neither hexasaccharides nor octasaccharides were able to stimulate receptor binding to the level attained by intact heparin. In fact, these oligosaccharides interfered with the ability of intact heparin in promoting FGF-receptor binding. The presence of both stimulatory and inhibitory activities in hexasaccharide and octasaccharide populations could be attributed to structural heterogeneity within the oligosaccharide preparations. However, similar observations were obtained with "highly-sulfated" structurally homogeneous preparations of hexasaccharide and octasaccharide, although these molecules generally had greater stimulatory and less inhibitory activity than their structurally heterogeneous counterparts. Hexasaccharides were found to be the smallest fragments able to potentiate the FGF-1-induced 3T3 cell proliferation while their effect on FGF-2 signaling was less clear. These observations suggest that heparin can modulate FGF-signaling at several stages with different end results.

3T3 Cells↗
PubMed1996

Enzymatic preparation of heparin oligosaccharides containing antithrombin III binding sites.

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are DeltaUAp2S(1-->4)-alpha-DGlcNpS6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D -GlcNpAc6S(1-->4)-betaD-GlcAp(1-->4)-alpha-D-GlcNpS 3S6S(1-->4)-alpha- L-IdoAp2S(1-->4)alpha-D-GlcNpS6S (where DeltaUAp is 4-deoxy-alpha-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp++ +(1-->4)-alpha- D-GlcNpAc6S (1-->4)-beta-D-GlcAp(1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha- L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha -D-GlcNpS 6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is DeltaUAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp++ +(1-->4)-alpha- D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpS6S . The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.

Animals↗
PubMed1996

NMR solution conformation of heparin-derived tetrasaccharide.

The solution conformation of the homogeneous, heparin-derived tetrasaccharide delta UA2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S (residues A, B, C and D respectively, where IdoA is iduronic acid) has been investigated by using 1H- and 13C-NMR. Ring conformations have been defined by J-coupling constants and inter-proton nuclear Overhauser effects (NOEs), and the orientation of one ring with respect to the other has been defined by inter-ring NOEs. NOE-based conformational modelling has been done by using the iterative relaxation matrix approach (IRMA), restrained molecular dynamics simulations and energy minimization to refine structures and to distinguish between minor structural differences and equilibria between various ring forms. Both glucosamine residues B and D are in the 4C1 chair conformation. The 6-O-sulphate group is oriented in the gauche-trans configuration in the D ring, whereas in the B ring the gauche-gauche rotomer predominates. Uronate (A) and iduronate (C) residues are mostly represented by 1H2 and 2S0 twisted boat forms, respectively, with small deviations in expected coupling constants and NOEs suggesting minor contributions from other A and C ring conformations.

Carbohydrate Conformation↗
PubMed1996

A new glycosaminoglycan from the giant African snail Achatina fulica.

A new glycosaminoglycan has been isolated from the giant African snail Achatina fulica. This polysaccharide had a molecular weight of 29,000, calculated based on the viscometry, and a uniform repeating disaccharide structure of -->4)-2-acetyl,2-deoxy-alpha-D-glucopyranose (1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->. This polysaccharide represents a new, previously undescribed glycosaminoglycan. It is related to the heparin and heparan sulfate families of glycosaminoglycans but is distinctly different from all known members of these classes of glycosaminoglycans. The structure of this polysaccharide, with adjacent N-acetylglucosamine and 2-sulfo-iduronic acid residues, also poses interesting questions about how it is made in light of our current understanding of the biosynthesis of heparin and heparan sulfate. This glycosaminoglycan represents 3-5% of the dry weight of this snail's soft body tissues, suggesting important biological roles for the survival of this organism, and may offer new means to control this pest. Snail glycosaminoglycan tightly binds divalent cations, such as copper(II), suggesting a primary role in metal uptake in the snail. Finally, this new polysaccharide might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of glycosaminoglycan biosynthesis and to the semisynthesis of new glycosaminoglycan analogs having important biological activities.

Animals↗
PubMed1996

13C-NMR relation study of heparin-disaccharide interactions with tripeptides GRG and GKG.

Heparin is a polydisperse sulphated copolymer consisting mostly of 1-->4 linked glucosamine and uronic acid residues, i.e. 2-deoxy-2-sulphamido-D-glucopyranose 6-sulphate and L-idopyranosyluronic acid 2-sulphate. 13C NMR has been used to study the interactions of heparinase-derived and purified heparin disaccharide with N- and C-terminally-blocked tripeptides GRG and GKG. Titration of the disaccharide with peptide indicates that GRG binds the disaccharide more strongly than does GKG, with interactions in either case being stronger at uronate ring positions. In the presence of GRG, a carboxylate pKa depression suggests electrostatic interactions between the arginine guanidinium group and the uronate carboxylate group. 13C relaxation data have been acquired for all disaccharide and peptide carbons in the presence and absence of GRG and GKG. 13C relaxation rates for the disaccharide are significantly faster in the presence of peptide, especially with GRG. Analysis of these relaxation data has been done in terms of molecular diffusion constants, D [symbol: see text] and D parallel, and an angle alpha between D parallel and a molecular frame defined by the moment of inertia tensor calculated for an internally rigid disaccharide. Disaccharide conformational space in these calculations has been sampled for both uronate half-chair forms (2H1 and 1H2) and over a range of glycosidic bond angles defined by motional order parameters and inter-residue nuclear Overhauser effects (+/- 30 degree from the average). In the absence of peptide, the ratio D [symbol: see text] /D parallel falls between 0.4 and 0.7; therefore molecular diffusion occurs preferentially about D parallel, which runs through both disaccharide rings. In the presence of peptide, D [symbol: see text] /D parallel is decreased, indicating that GRG is oriented along D parallel and proximal to the uronic acid ring. A model for this is shown.

Amino Acid Sequence↗
PubMed1996

Heparin structure and interactions with basic fibroblast growth factor.

Crystal structures of heparin-derived tetra- and hexasaccharides complexed with basic fibroblast growth factor (bFGF) were determined at resolutions of 1.9 and 2.2 angstroms, respectively. The heparin structure may be approximated as a helical polymer with a disaccharide rotation of 174 degrees and a translation of 8.6 angstroms along the helix axis. Both molecules bound similarly to a region of the bFGF surface containing residues asparagine-28, arginine-121, lysine-126, and glutamine-135, the hexasaccharide also interacted with an additional binding site formed by lysine-27, asparagine-102, and lysine-136. No significant conformational change in bFGF occurred upon heparin oligosaccharide binding, which suggests that heparin primarily serves to juxtapose components of the FGF signal transduction pathway.

Binding Sites↗