We have demonstrated by affinity chromatography that hepatocyte growth factor/scatter factor (HGF/SF) binds strongly to dermatan sulfate (DS), with a similar ionic strength dependence to that previously seen with heparan sulfate (HS). Analysis of binding kinetics on a biosensor yields an equilibrium dissociation constant, K D , of 19.7 nM. This corresponds to a 10 -100-fold weaker interaction than that with HS, primarily due to a faster dissociation rate of the complex. The smallest DS oligosaccharide with significant affinity for HGF/SF by affinity chromatography appears to be an octasaccharide. A sequence comprising unsulfated iduronate residues in combination with 4-O-sulfated N-acetylgalactosamine is sufficient for high affinity binding. The presence of 2-Osulfation on the iduronate residues does not appear to be inhibitory. These observations concur with our previous suggestions, from analyses of HS binding (Lyon, M., Deakin, J. A., Mizuno, K., Nakamura, T., and Gallagher, J.T. (1994) J. Biol. Chem. 269, 11216 -11223), that N-sulfation of hexosamines and 2-O-sulfation of iduronates are not absolute requirements for glycosaminoglycan binding to HGF/SF. This is the first described example of a high affinity interaction between a growth factor and DS, and is likely to have significant implications for the biological activity of this paracrine-acting factor.Hepatocyte growth factor/scatter factor (HGF/SF) 1 is a pleiotropic factor with the ability to influence the growth, motility, differentiation, and morphogenesis of its target cells (for a recent review, see Ref. 1). It acts in a paracrine manner, with the major secretors being fibroblasts, vascular smooth muscle cells, nonparenchymal liver cells, etc., whereas those cells that possess the requisite tyrosine kinase receptor (Met) are primarily epithelial and endothelial cells. Recent evidence suggests that multipotent and erythroid hemopoietic progenitor cells are also responsive to HGF/SF. The HGF/SF-Met system appears to operate primarily during the morphogenetic and differentiation events occurring in organogenesis, as well as in the repair of organ damage in the adult (reviewed in Ref. 1). Aberrant expression of HGF/SF and/or Met has been strongly implicated in tumor progression, particularly in the acquisition of an invasive malignant phenotype (2-5). This presumably results from its ability to directly stimulate the growth and motility of tumor cells, as well as increasing the secretion of matrix-degrading proteases (6), thereby facilitating invasion of the surrounding stroma. Additionally, its potent angiogenic action (7, 8) may contribute to the development of a tumor vasculature, which is essential for sustaining an expanding tumor mass.In addition to Met, HGF/SF also interacts in vitro with the heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) (9). The latter probably constitute the more abundant, but relatively lower affinity, HGF/SF-binding sites present on most cells (10). The interaction of HGF/SF with cell surface HSPGs may ...

The heparan sulfates (HS) are hypervariable linear polysaccharides that act as membrane co-receptors for growth factors, chemokines, and extracellular matrix proteins. In most instances, the molecular basis of protein recognition by HS is poorly understood. We have sequenced 75% of the sulfated domains (S-domains) of fibroblast HS, including all of the major ones. This analysis revealed tight coupling of N-and 2-O-sulfation and a low frequency but precise positioning of 6-O-sulfates, which are required functional groups for HS-mediated activation of the fibroblast growth factors. S-domain sequencing was conducted using a novel and highly sensitive method based on a new way of reading the sequence from high performance liquid chromatography separation profiles of metabolically labeled HS-saccharides following specific chemical and enzymatic scission. The implications of the patterns seen in the sulfated domains for better understanding of the synthesis and function of HS are discussed.Many biological macromolecules have information encoded in their primary structure. In proteins, the amino acid sequence determines the secondary and tertiary structural characteristics of the folded protein, whereas in DNA and RNA the nucleotide sequence is the means of storing coded information that can be read by the transcriptional and translational machinery of the cell. Information for ligand binding and activation is also present within the structure of complex saccharides (1, 2). Heparan sulfate (HS) 1 is one of a class of polysaccharides known as glycosamino glycans, and recent research indicates that it expresses important protein recognition domains within its primary sequence (3, 4). However, unlike nucleic acids and protein, there is no established method available to read this primary information.The near ubiquitous occurrence and strategic positioning of heparan sulfate proteoglycans, both at the surface of most mammalian cells and in the extracellular matrix, is a good indicator of their role in cell-cell recognition and cell-matrix adhesion (5, 6). Heparan sulfate proteoglycans are key sites of interaction and signaling when cells form focal adhesions on extracellular matrix substrates such as fibronectin (7). A very extensive range of growth factors and cytokines, including key angiogenic factors such as basic fibroblast growth factor (bFGF) (8, 9), hepatocyte growth factor (10), and vascular endothelial growth factor (11), have been shown to not only bind HS in vitro but to require its presence as a membrane coreceptor for optimal activation of their cognate, high affinity signaling receptors. The similarity between the co-receptor role of HS and the effect of heparin in facilitating the inactivation of thrombin by antithrombin is indicative of many biological processes being driven by HS catalysis. It may therefore be possible to exploit an understanding of HS-growth factor interactions to modulate the effects of these ligands (12) in a similar way to the current clinical use of heparin. Although there is a sub...

A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His Age-related macular degeneration (AMD) 2 is a leading cause of irreversible visual impairment in the elderly (1). The densely packed photoreceptors in the macula are maintained by the underlying retinal pigment epithelium (RPE) (2). A unique pentalaminar extracellular matrix (ECM), the Brüch's membrane, separates the RPE from the fenestrated endothelium of the choroidal vasculature. In early AMD, fatty deposits, called drusen, appear between Brüch's membrane and the RPE (3). Early AMD may progress to severe forms (4), characterized by RPEcell death and atrophy of the photoreceptor layer, or choroidal neovascularization.Drusen are rich in cell breakdown products and proteins of the complement system (5). Complement (6, 7) is a potent mediator of inflammation. An association between AMD and the gene (CFH) for the complement regulator factor H (fH) was demonstrated by Hageman et al. (8) and confirmed independently by others (9 -12). Hageman et al. (8) additionally reported the RPE to be a source of fH, while drusen components C3a and C5a have been implicated in neovascularization in mouse models of AMD (13), strengthening further the evidence for an AMD-complement link.The at-risk allele that has received most attention, in CFH exon 9 (rs1061170; 1277T Ͼ C), encodes a His rather than a Tyr at position 402 (residue 384 of the mature fH) and is present in ϳ35% of individuals of European descent. Homozygous individuals have a 6-fold increased risk of developing AMD, whereas heterozygotes are 2.5 times more susceptible (14). Doubts over a causal link between the H402Y variation and the etiology of AMD have been raised by the identification of 20 synonymous or intronic single nucleotide polymorphisms (SNPs) in a 123-kb region overlapping CFH that are even more strongly associated with AMD (15, 16). Moreover, variations in genes encoding complement factor 2 (C2) and complement factor B (BF) also strongly influence risk, as does an SNP in a gene, LOC387715, of unknown function (16,17). Other risk factors