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

Sulfated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns/densities of sulfation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulfate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analyzing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving prederivatization of disaccharides with 2-aminoacridone with no cleanup of samples, followed by a separation by reverse-phase HPLC that is sensitive to as little as approximately 100 pg (approximately 10(-13) mol) of an individual disaccharide, thereby allowing analyses of >10 ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed glycosaminoglycan pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool) and MDCK cells (which revealed a HS species of relatively low N-sulfation, but high O-sulfation). This simplified method should find a widespread utility for analyzing glycosaminoglycans from limited animal and cell culture samples.

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Hepatocyte growth factor/scatter factor, in addition to binding to its specific signal-transducing receptor, Met, also interacts with both heparan and dermatan sulfates with high affinity. We have investigated the comparative role of these two glycosaminoglycans in the activation of Met by hepatocyte growth factor/scatter factor. Using glycosaminoglycan-deficient CHO pgsA-745 cells we have shown that growth factor activity is critically dependent upon glycosaminoglycans, and that heparan sulfate and dermatan sulfate are equally potent as coreceptors. Cross-linked 1:1 conjugates of growth factor and either heparan or dermatan sulfate do not dimerize under physiological conditions and are biologically active. This implies that a ternary signaling complex with Met forms in vivo. Native Met isolated from CHO pgsA-745 cells shows only very weak intrinsic affinity for heparin in vitro. Also, a heparin-derived hexasaccharide, which is the minimal size for high affinity binding to the growth factor alone, is sufficient to induce biological activity. Together these observations imply that the role of these glycosaminoglycan may be primarily to effect a conformational change in hepatocyte growth factor/ scatter factor, rather than to induce a necessary growth factor dimerization, or to stabilize a ternary complex by additionally interacting with Met.

Hepatocyte growth factor/scatter factor (HGF/SF) has a cofactor requirement for heparan sulfate (HS) and dermatan sulfate (DS) in the optimal activation of its signaling receptor MET. However, these two glycosaminoglycans (GAGs) have different sugar backbones and sulfation patterns, with only the presence of iduronate in common. The structural basis for GAG recognition and activation is thus very unclear. We have clarified this by testing a wide array of natural and modified GAGs for both protein binding and activation. Comparisons between Ascidia nigra (2,6-O-sulfated) and mammalian (mainly 4-O-sulfated) DS species, as well as between a panel of specifically desulfated heparins, revealed that no specific sulfate isomer, in either GAG, is vital for interaction and activity. Moreover, different GAGs of similar sulfate density had comparable properties, although affinity and potency notably increase with increasing sulfate density. The weaker interaction with CS-E, compared with DS, shows that GlcA-containing polymers can bind, if highly sulfated, but emphasizes the importance of the flexible IdoA ring. Our data indicate that the preferred binding sites in DS in vivo will be comprised of disulfated, IdoA(2S)-containing motifs. In HS, clustering of N-/2-O-/6-O-sulfation in S-domains will lead to strong reactivity, although binding can also be mediated by the transition zones where sulfates are mainly at the N-and 6-O-positions. GAG recognition of HGF/SF thus appears to be primarily driven by electrostatic interactions and exhibits an interesting interplay between requirements for iduronate and sulfate density that may reflect in part a preference for particular sugar chain conformations.

Full-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/ dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.