Jonathan R. Fromm

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° 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.

Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins

Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein‐GAG binding have not been well‐characterised. Ionic interactions are important in promoting protein‐GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein‐GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 Å spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein‐GAG interactions play a prominent role in cell‐cell interaction and cell growth. Pathogens including virus particles might target GAG‐binding sites in envelope proteins leading to infection. BioEssays 20:156–167, 1998.

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.

Importance of specific amino acids in protein binding sites for heparin and heparan sulfate

Heparin and heparan sulfate bind a variety of proteins and peptides to regulate many biological activities. Past studies have examined a limited number of established heparin binding sites and have focused on basic amino acids when modeling binding site structural motifs. This study examines the prevalence of individual amino acids in peptides binding to heparin or heparan sulfate. A 7-mer random peptide library was synthesized using the 20 common amino acids. This 7-mer library was affinity separated using both heparin and heparan sulfate-Sepharose. Bound peptide populations were eluted with a salt step gradient (pH 7) and analysed for amino acid composition. Peptides released from heparin-Sepharose by 0.3 M NaCl were enriched in arginine, lysine, glycine and serine; and depleted in methionine and phenylalanine. In contrast, peptides released from heparan sulfate-Sepharose were enriched in arginine, glycine, serine, and proline (at 0.15 M NaCl). These peptides were depleted in histidine, isoleucine, methionine (not detectable) and phenylalanine. In the heparin binding sites of proteins, which have been published, the enriched amino acids were arginine, lysine and tyrosine. Depleted amino acids include aspartic acid, glutamic acid, glutamine, alanine, glycine, phenylalanine, serine, threonine and valine. This study demonstrates that heparin and heparan sulfate bind different populations of peptide sequences. The differences in amino acid composition indicate that the positive charge density and spacing requirements differ for peptides binding these two glycosaminoglycans.

Lectin affinity electrophoresis

Lectin affinity electrophoresis is a powerful technique to investigate the interaction between a lectin and its ligand. Affinity electrophoresis results from the reduced mobility of a charged species owing to its interaction with an immobile species. In this protocol, a two-dimensional lectin affinity electrophoresis experiment is described that affords separation of oligosaccharides. The first-dimension is composed of a weak, polyacrylamide, capillary tube gel containing a lectin. The example described involves a mixture of fluorescently labeled disaccharides. The mobility of only the lectin-binding disaccharide is reduced affording a separation in the first-dimension. The tube gel is then extruded and placed onto the second-dimension gradient polyacrylamide gel and subjected to electrophoresis. Mobility in the second-dimension is dependent on molecular size and visualization is by fluorescence under transillumination. This method is also applicable, with appropriate modifications, for the separation and analysis of glycopeptides and glycoproteins.

GLYCOPROTEIN C(gC) of HERPES SIMPLEX VIRUS (HSV) TYPE 1 BINDS TWO DISTINCT POLYSACCHARIDE POPULATIONS WITHIN HEPARIN. † 704

GLYCOPROTEIN C(gC) of HERPES SIMPLEX VIRUS (HSV) TYPE 1 BINDS TWO DISTINCT POLYSACCHARIDE POPULATIONS WITHIN HEPARIN. † 704

GLYCOPROTEIN C OF HERPES SIMPLEX VIRUS TYPE 1 BINDS TO SPECIFIC POLYSACCHARIDES WITHIN HEPARIN. 1016

GLYCOPROTEIN C OF HERPES SIMPLEX VIRUS TYPE 1 BINDS TO SPECIFIC POLYSACCHARIDES WITHIN HEPARIN. 1016