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R J Linhardt

Publications and source records attributed to R J Linhardt.

At least 19 recordsLinked to original sources
PubMed2006

Synthetic oligosaccharide stimulates and stabilizes angiogenesis: structure-function relationships and potential mechanisms.

To determine the proangiogenesis effect of series of saccharides and a synthetic oligosaccharide and potential mechanisms, an in vitro 3-dimensional endothelial cell sprouting (3D-ECS) assay and the chick chorioallantoic membrane (CAM) model were used. We demonstrated that a sulfated oligosaccharide significantly promotes the endothelial capillary network initiated by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF). Furthermore, although the capillary network initiated by VEGF and b-FGF lasts no more than 7 days, addition of a sulfated oligosaccharide significantly amplifies angiogenesis and stabilizes the capillary network of new blood vessels. In the CAM model, sulfated oligosaccharide also stimulated angiogenesis. In both the CAM and the 3D-ECS assay, structure-function studies reveal that increased saccharide chain length up to the hexa- to decasaccharide show optimal proangiogenesis efficacy. In addition, the sulfation and molecular shape (branched vs linear) of oligosaccharide are important for sustained proangiogenesis efficacy. Data indicate that chemically defined synthetic oligosaccharides can play an important role in regulation of capillary structure and stability, which may contribute to future advances in therapeutic angiogenesis. The proangiogenesis efficacy of an oligosaccharide is mediated via integrin alphavbeta3 and involves mitogen-activated protein kinase signaling mechanisms.

Animals↗
PubMed2003

Distribution of N-acetylneuraminic acid and sialylglycan in eggs of the Silky fowl.

1. The distribution of sialic acid in the eggs of original Silky fowl was investigated. The sialic acid contents of the yolk, albumen and the chalaza of a single egg were 205.2, 11.96 and 0.83 mg, respectively. 2. The sialic acid content of the yolk of Silky eggs was 11.5-fold higher than that of a conventional domestic fowl yolk. 3. Sialic acid isolated from Silky yolk was entirely N-acetylneuraminic acid (NeuAc). No N-glycolylneuraminic acid or O-acetyl containing sialic acid was observed. 4. The structure of the major sialylglycan in Silky egg yolk was determined to be a disialyl-biantennary chain in which the NeuAc residues were alpha2-6 linked to glucose. No alpha2-3 linkage was observed. 5. Thus, the Silky fowl's egg provides an excellent source of NeuAc and sialylglycan.

Animals↗
PubMed2001

Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with different heparan sulfate motifs.

Stromal cell-derived FGF-7 binds and activates only the resident FGFR2IIIb in epithelial cells while FGF-1 and FGF-2 exhibit a broader interaction with multiple isoforms of FGFR. Here we report the structure of FGF-7 that has been solved to 3.1 A resolution by molecular replacement with the structure of a dual function chimera of FGF-7 and FGF-1 (FGF-7/1) which was resolved to 2.3 A. Comparison of the FGF-7 structure to that of FGF-1 and FGF-2 revealed the strongly conserved Calpha backbone among the three FGF polypeptides and the surface hydrophobic patch that forms the primary receptor-binding domain. In contrast, a decrease and dispersion of the positive surface charge density characterized the heparin-binding domain of FGF-7 defined by homology to that of FGF-1 and FGF-2 in complexes with heparin. A simple heparin hexasaccharide that cocrystallized with FGF-1 and FGF-2 and protected both against protease in solution failed to exhibit the same properties with FGF-7. In contrast to FGF-1 and FGF-2, protection of FGF-7 was enhanced by heparin oligosaccharides of increased length with those exhibiting a 3-O-sulfate being the most effective. Protection of FGF-7 required interaction with specifically the fraction of crude heparin retained on antithrombin affinity columns. Conversely, heparin enriched by affinity for immobilized FGF-7 exhibited anti-factor Xa activity similar to that purified on an antithrombin affinity matrix. In contrast, an FGF-1 affinity matrix enriched the fraction of crude heparin with low anti-factor Xa activity. The results provide a structural basis to suggest that the unique FGF-7 heparin-binding (HB) domain underlies a specific restriction in respect to composition and length of the heparan sulfate motif that may impact specificity of localization, stability, and trafficking of FGF-7 in the microenvironment, and formation and activation of the FGFR2IIIb kinase signaling complex in epithelial cells.

Amino Acid Sequence↗
PubMed2001

Direct measurement of the interactions of glycosaminoglycans and a heparin decasaccharide with the malaria circumsporozoite protein.

Circumsporozoite (CS) protein is a predominant surface antigen of malaria sporozoites, the infective form of the parasite, and has been used for making anti-malaria vaccines. For the first time we have examined the interaction of CS protein with various glycosaminoglycans in real time using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Heparin was the best binder among the glycosaminoglycans tested and bound to CS protein with nanomolar affinity. Using purified and structurally defined small heparin oligosaccharides, we identified a decasaccharide to be the minimum sized CS protein-binding sequence. In an indirect competition assay, this decasaccharide blocked the CS protein interaction with HepG2 cells with an ID(50) of less than 60 nM. The decasaccharide has a structure commonly found in hepatic heparan sulfate, and the same sequence has recently been shown to bind specifically to apolipoprotein E. Examination of porcine liver heparan sulfate in this indirect competition assay showed that it and heparin were the only glycosaminoglycans that could effectively block CS protein interaction with HepG2 cells in culture. These data support the hypothesis that the invasion of liver cells by the parasite shares a common mechanism with the hepatic uptake of lipoprotein remnants from the blood.

Animals↗
PubMed2001

New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E.

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, (13)C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the alpha-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.

Animals↗
PubMed2001

Certain high molecular weight heparin chains have high affinity for vitronectin.

Vitronectin is a 70-kDa protein that is found in both the extracellular matrix as well as serum. Vitronectin is one of the few proteins that regulates both the complement and the coagulation systems. Heparin is known to bind to vitronectin. Review of the literature reveals apparently conflicting outcomes of the interaction of heparin, vitronectin, and the complement system. Previous studies demonstrated that heparin diminishes vitronectin inhibition of complement activity. Numerous studies have also demonstrated that heparin exerts a net inhibitory effect on complement. We used two dimensional affinity resolution electrophoresis (2DARE) to examine this apparent paradox. 2DARE allowed simultaneous determination of binding affinity of heparin for vitronectin as well as the M(r) of the heparin species. In the 2DARE experiment, the interaction of heparin with vitronectin caused retardation of the movement of the heparin through the tube gel in the first dimension. The degree of the retardation of movement was used to calculate the approximate K(d) of that interaction. The heparin from the tube gel was then subjected to a second dimension electrophoresis to determine the M(r) of the heparin. 2DARE analysis of the interaction of heparin with vitronectin clearly demonstrated that a sub-population of heparin chains with M(r) > 8000 bound vitronectin with high affinity whereas most high M(r) chains and all lower M(r) chains showed little to no affinity for vitronectin. Our findings are consistent with the hypothesis that a unique binding domain exists in certain heparin chains for vitronectin.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Probing the interaction of dengue virus envelope protein with heparin: assessment of glycosaminoglycan-derived inhibitors.

A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.

Anticoagulants↗
PubMed2001

Interaction of the N-terminal domain of apolipoprotein E4 with heparin.

Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimer's disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->[4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->](3)4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.

Animals↗
PubMed2001

Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis.

The crystal structures of Flavobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide (HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively. The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution. For each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel. We suggest that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area. The putative catalytic site is located between the substrate recognition area and the product release area, carrying out catalysis at the +1 subsite. Four residues near the catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic tetrad. The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg292Ala mutant. Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis. Candidates for the general base, removing the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or His225. Tyrosine 234 is a candidate to protonate the leaving group. Arginine 288 likely contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis.

Amino Acid Sequence↗
PubMed2001

Annexin V--heparin oligosaccharide complex suggests heparan sulfate--mediated assembly on cell surfaces.

BACKGROUND: Annexin V, an abundant anticoagulant protein, has been proposed to exert its effects by self-assembling into highly ordered arrays on phospholipid membranes to form a protective anti-thrombotic shield at the cell surface. The protein exhibits very high-affinity calcium-dependent interactions with acidic phospholipid membranes, as well as specific binding to glycosaminoglycans (GAGs) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. At present, there is no structural information to elucidate this interaction or the role it may play in annexin V function at the cell surface. RESULTS: We report the 1.9 A crystal structure of annexin V in complex with heparin-derived tetrasaccharides. This structure represents the first of a heparin oligosaccharide binding to a protein where calcium ions are essential for the interaction. Two distinct GAG binding sites are situated on opposite protein surfaces. Basic residues at each site were identified from the structure and site-directed mutants were prepared. The heparin binding properties of these mutants were measured by surface plasmon resonance. The results confirm the roles of these mutated residues in heparin binding, and the kinetic and thermodynamic data define the functionally distinct character of each distal binding surface. CONCLUSION: The annexin V molecule, as it self-assembles into an organized array on the membrane surface, can bind the heparan sulfate components of cell surface proteoglycans. A novel model is presented in which proteoglycan heparan sulfate could assist in the localization of annexin V to the cell surface membrane and/or stabilization of the entire molecular assembly to promote anticoagulation.

Animals↗
PubMed2001

Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization.

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.

Animals↗
PubMed2001

Order out of complexity--protein structures that interact with heparin.

Many proteins of widely differing functionality and structure are capable of binding heparin. Structural characterisations of the many types of such complexes are being reported in ever-increasing number and at improved resolution. Several crystal structures of complexes formed through the interaction of heparin-derived oligosaccharides with one or more protein partners have been described.

Annexin A5↗
PubMed2001

Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica.

Acharan sulfate is a glycosaminoglycan (GAG), having the structure -->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose(1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3-5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.

Alcian Blue↗
PubMed2001

Anticoagulant and antiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate (PI-88).

Heparinomimetic mannopentaose phosphate sulfate (PI-88) (Progen Industries Ltd. Brisbane, Australia), currently developed as an anticoagulant and antiproliferative agent, mainly is composed of a pentomannan. However, tetrasaccharide and disaccharide components are also present. The molecular profile and the anticoagulant potency of PI-88 are investigated in this study. Gel permeation chromatography and polyacrylamide gel electrophoresis analyses were carried out to determine the molecular profile and separation of components of PI-88, respectively. Potentiation of antithrombin III (ATIII) and heparin cofactor-II (HC-II) activity were measured using chromogenic substrate assay. In order to determine anticoagulant and antiprotease effects of PI-88, various global anticoagulant tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Hep-test (Haemachem Inc., St. Louis), ecarin clotting time (ECT), activated clotting time (ACT), and thromboelastography (TEG) were used. Anti-Xa and anti-IIa activities also were measured. The effect of PI-88 on the release of tissue factor pathway inhibitor (TFPI) was performed in nonhuman primates who received PI-88 and in endothelial cell culture systems. The relative susceptibility of PI-88 to heparinase I, protamine sulfate (PS), and platelet factor 4 (PF4) also was evaluated. The high-performance liquid chromatography profiles of PI-88 showed that its average molecular weight is approximately 2300 Da. Separation and gradient electrophoretic patterns of PI-88 showed that it is composed of five different fractions. This agent activates HC-II through inhibiting the thrombin generation but not inhibiting ATIII. Although PI-88 produced a concentration-dependent prolongation of all of the clotting tests, ECT gave the best correlation in the dose-response curve (ECT, r2 = 0.94; TT, r2 = 0.84; APTT, r2 = 0.69). Heparinomimetic mannopentaose phosphate sulfate (PI-88) exhibited marked inhibition of FIIa, but not of FXa. Heparinase I failed to produce significant neutralization of PI-88 in all the assays used, whereas PS and PF4 partially neutralized the effects of this compound. Heparinomimetic mannopentaose phosphate sulfate (PI-88) produced fivefold increase in the TFPI levels at 15 minutes after intravenous (IV) injection to primates. The incubation of PI-88 in endothelial cell culture system also showed a strong effect on TFPI release. These results suggest that PI-88 exhibited strong antithrombotic and anticoagulant activity in addition to its known antiproliferative properties. Because of the molecular characteristics and the dual nature of the pharmacologic action of PI-88, this agent represents an attractive pharmacologic agent for the control of thrombotic and proliferative disorders.

Anticoagulants↗
PubMed2001

Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.

Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.

Alkaline Phosphatase↗