Complement Factor H Binds at Two Independent Sites to C-reactive Protein in Acute Phase Concentrations

Factor H (FH) regulates the activation of C3b in the alternative complement pathway, both in serum and at host cell surfaces. It is composed of 20 short complement regulator (SCR) domains. The Y402H polymorphism in FH is a risk factor for age-related macular degeneration. C-reactive protein (CRP) is an acute phase protein that binds Ca²⁺. We established the FH-CRP interaction using improved analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and synchrotron x-ray scattering methods. Physiological FH and CRP concentrations were used in 137 mm NaCl and 2 mm Ca²⁺, in which the occurrence of denatured CRP was avoided. In solution, AUC revealed FH-CRP binding. The FH-CRP interaction inhibited the formation of higher FH oligomers, indicating that CRP blocked FH dimerization sites at both SCR-6/8 and SCR-16/20. SPR confirmed the FH-CRP interaction and its NaCl concentration dependence upon using either immobilized FH or CRP. The SCR-1/5 fragment of FH did not bind to CRP. In order of increasing affinity, SCR-16/20, SCR-6/8 (His-402), and SCR-6/8 (Tyr-402) fragments bound to CRP. X-ray scattering showed that FH became more compact when binding to CRP, which is consistent with CRP binding at two different FH sites. We concluded that FH and CRP bind at elevated acute phase concentrations of CRP in physiological buffer. The SCR-16/20 site is novel and indicates the importance of the FH-CRP interaction for both age-related macular degeneration and atypical hemolytic uremic syndrome.     

Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein-protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD.     

Multimeric Interactions between Complement Factor H and Its C3d Ligand Provide New Insight on Complement Regulation

Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations. Surface plasmon resonance studies showed that native FH binds C3d in two stages. An equilibrium dissociation constant K_D1 of 2.6 μM in physiological buffer was determined for the first stage. Overlay experiments indicated that C3d formed multimeric complexes with FH. X-ray scattering showed that the maximum dimension of the C3d complexes with SCR-16/20 at 29 nm was not much longer than that of the unbound SCR-16/20 dimer. Molecular modelling suggested that the ultracentrifugation and scattering data are most simply explained in terms of associating dimers of each of SCR-16/20 and C3d. We conclude that the physiological interaction between FH and C3d is not a simple 1:1 binding stoichiometry between the two proteins that is often assumed. Because the multimers involve the C-terminus of FH, which is bound to host cell surfaces, our results provide new insight on FH regulation during excessive complement activation, both in the fluid phase and at host cell surfaces decorated by C3d.     

The structural plasticity of heparan sulfate NA-domains and hence their role in mediating multivalent interactions is confirmed by high-accuracy <Superscript>15</Superscript>N-NMR relaxation studies

Considering the biological importance of heparan sulfate (HS) and the significant activity of its highly-sulfated regions (S-domains), the paucity of known functions for the non-sulfated NA-domains is somewhat puzzling. It has been suggested that chain dynamics within the NA-domains are the key to their functional role in HS. In this study, we investigate this hypothesis using state-of-the-art nuclear magnetic resonance (NMR) experiments at multiple frequencies. To resolve the problem of severe overlap in (1)H-NMR spectra of repetitive polysaccharides from proteoglycans, we have prepared oligosaccharides with the chemical structure of HS NA-domains containing the (15)N nucleus, which has enough chemical shift dispersion to probe the central residues of octasaccharides at atomic resolution using 600 MHz NMR. By performing NMR relaxation experiments at three magnetic-field strengths, high quality data on internal dynamics and rotational diffusion was obtained. Furthermore, translational diffusion could also be measured by NMR using pulse field gradients. These experimental data were used, in concert with molecular dynamics simulations, to provide information on local molecular shape, greatly aiding our relaxation analyses. Our results, which are more accurate than those presented previously, confirm the higher flexibility of the NA-domains as compared with reported data on S-domains. It is proposed that this flexibility has two functional roles. First, it confers a greater area of interaction from the anchoring point on the core protein for the bioactive S-domains. Secondly, it allows multiple interactions along the same HS chain that are dynamically independent of each other.     

Avidin is a heparin-binding protein. Affinity,specificity and structural analysis

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.     

Associative and structural properties of the region of complement factor H encompassing the Tyr402His disease-related polymorphism and its interactions with heparin

Factor H (FH) is a major complement control protein in serum. The seventh short complement regulator (SCR-7) domain of the 20 in FH is associated with age-related macular degeneration through a Tyr402His polymorphism. The recombinant SCR-6/8 domains containing either His402 or Tyr402 and their complexes with a heparin decasaccharide were studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient is concentration dependent, giving a value of 2.0 S at zero concentration and a frictional ratio f/f(o) of 1.2 for both allotypes. The His402 allotype showed a slightly greater self-association than the Tyr402 allotype, and small amounts of dimeric SCR-6/8 were found for both allotypes in 50 mM, 137 mM and 250 mM NaCl buffers. Sedimentation equilibrium data were interpreted in terms of a monomer-dimer equilibrium with a dissociation constant of 40 microM for the His402 form. The Guinier radius of gyration R(G) of 3.1-3.3 nm and the R(G)/R(O) ratio of 2.0-2.1 showed that SCR-6/8 is relatively extended in solution. The distance distribution function P(r) showed a maximum dimension of 10 nm, which is less than the length expected for a linear domain arrangement. The constrained scattering and sedimentation modelling of FH SCR-6/8 showed that bent SCR arrangements fit the data better than linear arrangements. Previously identified heparin-binding residues were exposed on the outside curvature of this bent domain structure. Heparin caused the formation of a more linear structure, possibly by binding to residues in the linker. It was concluded that the His402 allotype may self-associate more readily than the Tyr402 allotype, SCR-6/8 is partly responsible for the folded-back structure of intact FH, and SCR-6/8 changes conformation upon heparin binding.     

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.     

Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46

NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.     

The regulatory SCR-1/5 and cell surface-binding SCR-16/20 fragments of factor H reveal partially folded-back solution structures and different self-associative properties

Factor H (FH) is a plasma glycoprotein that plays a central role in regulation of the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and heparin. X-ray scattering and analytical ultracentrifugation showed that SCR-1/5 was monomeric, while SCR-16/20 formed dimers. The Guinier radius of gyration R_G of 4.3 nm for SCR-1/5 and those of 4.7 nm and about 7.8 nm for monomeric and dimeric SCR-16/20, respectively, showed that their structures are partially folded back and bent. The distance distribution function P(r) showed that SCR-1/5 has a maximum dimension of 15 nm while monomeric and dimeric SCR-16/20 are 17 nm and about 27 nm long, respectively. The sedimentation coefficient of 2.4 S for SCR-1/5 showed no concentration-dependence, while that for SCR-16/20 was 2.8 S for the monomer and 3.9 S for the dimer. Sedimentation equilibrium data showed that SCR-1/5 is monomeric while SCR-16/20 exhibited a weak monomer-dimer equilibrium with a dissociation constant of 16 μM. The constrained scattering and sedimentation modelling of SCR-1/5 and SCR-16/20 showed that partially folded-back and bent flexible SCR arrangements fitted both data sets better than extended linear arrangements, and that the dimer was best modelled in the SCR-16/20 model by an end-to-end association of two SCR-20 domains. The SCR-1/5 and SCR-16/20 models were conformationally similar to the previously determined partially folded-back structure for intact wild-type FH, hence suggesting a partial explanation of the intact FH structure. Comparison of the SCR-16/20 model with the crystal structure of C3b clarified reasons for the distribution of mutations leading to atypical haemolytic uraemic syndrome.     

Binding of interferon-gamma to heparan sulfate is restricted to the heparin-like domains and involves carboxylic--but not N-sulfated--groups.

Interferon-gamma binds to the glycosaminoglycan part of basement membrane proteoglycan. To obtain a greater insight into this interaction, different glycosaminoglycans and their subfractions were used in various binding assays. High affinity binding occurs with heparin and heparan sulfate only, the latter being the predominant basement membrane glycosaminoglycan. Furthermore, using heparan sulfate and heparin treated with heparinases I and III, we have shown that the interferon-gamma binding sites are localized on the N-sulfated glucosamine rich domains of the molecule. Interestingly, interferon-gamma and fibroblast growth factor compete for the same binding domain on heparan sulfate, although they are unrelated proteins. This last point is discussed in the light of the conformational flexibility of the glycosaminoglycan molecules.     

Heparan sulfate primed on β-D-xylosides restores binding of basic fibroblast growth factor

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA-745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure of pgsA-745 cells to β-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell sulfate. Restoration of bFGF binding correlated with the ability of the β-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10–30 μM) of estradiol-β-D-xyloside and naphthyl-β-D-xyloside, but not by cis/trans-decahydro-2-naphthyl-β-D-xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5–1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors.     

The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.     

Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore 2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and exosequencing of oligosaccharides

A series of disaccharides derived from chondroitin sulfate and heparin/heparan sulfate were derivatized at their reducing ends with a fluorophore 2-aminobenzamide to develop a sensitive microanalytical method for glycosaminoglycans. The resulting labeled compounds derived from chondroitin sulfate or heparin/heparan sulfate were well-separated and quantified by HPLC equipped with a fluorescence detector. The detection limit was a low picomole level. This method was applied to the analysis of the disaccharide composition of tetra- and hexasaccharides derived from chondroitin sulfate and heparin/heparan sulfate as well as these glycosaminoglycan polysaccharides. The method was also successfully applied to the exosequencing of chondrohexasaccharides, where the fluorophore-labeled oligosaccharides were degraded exolytically from the nonreducing ends using bacterial eliminases. The resultant labeled fragments were identified by HPLC.     

Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19–20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19–20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19–20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19–20 binding to mGEnCs. Compared with wild type FH19–20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19–20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19–20 mutants.     

Folded-back solution structure of monomeric factor H of human complement by synchrotron X-ray and neutron scattering, analytical ultracentrifugation and constrained molecular modelling

Factor H (FH) is a regulatory cofactor for the protease factor I in the breakdown of C3b in the complement system of immune defence, and binds to heparin and other polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains, for which the overall arrangement in solution is unknown. As previous studies had shown that FH can form monomeric or dimeric structures, X-ray and neutron scattering was accordingly performed with FH in the concentration range between 0.7 and 14 mg ml(-1). The radius of gyration of FH was determined to be 11.1-11.3 nm by both methods, and the radii of gyration of the cross-section were 4.4 nm and 1.7 nm. The distance distribution function P(r) showed that the overall length of FH was 38 nm. The neutron data showed that FH was monomeric with a molecular mass of 165,000(+/-17,000) Da. Analytical ultracentrifugation data confirmed this, where sedimentation equilibrium curve fits gave a mean molecular mass of 155,000(+/-3,000) Da. Sedimentation velocity experiments using the g*(s) derivative method showed that FH was monodisperse and had a sedimentation coefficient of 5.3(+/-0.1) S. In order to construct a full model of FH for scattering curve and sedimentation coefficient fits, homology models were constructed for 17 of the 20 SCR domains using knowledge of the NMR structures for FH SCR-5, SCR-15 and SCR-16, and vaccinia coat protein SCR-3 and SCR-4. Molecular dynamics simulations were used to generate a large conformational library for each of the 19 SCR-SCR linker peptides. Peptides from these libraries were combined with the 20 SCR structures in order to generate stereochemically complete models for the FH structure. Using an automated constrained fit procedure, the analysis of 16,752 possible FH models showed that only those models in which the 20 SCR domains were bent back upon themselves were able to account for the scattering and sedimentation data. The best-fit models showed that FH had an overall length of 38 nm and is flexible. This length is significantly less than a predicted length of 73 nm if the 20 SCR structures had been arranged in an extended arrangement. This outcome is attributed to several long linker sequences. These bent-back domain structures may correspond to conformational flexibility in FH and enable the multiple FH binding sites for C3 and heparin to come into close proximity.     

Fibrillin-1 interactions with heparin. Implications for microfibril and elastic fiber assembly

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.     

Heparan sulfate regulates fibrillin-1 N- and C-terminal interactions

Fibrillin-1 N- and C-terminal heparin binding sites have been characterized. An unprocessed monomeric N-terminal fragment (PF1) induced a very high heparin binding response, indicating heparin-mediated multimerization. Using PF1 deletion and short fragments, a heparin binding site was localized within the domain encoded by exon 7 after the first hybrid domain. Rodent embryonic fibroblasts adhered to PF1 and deletion fragments, and, when cells were plated on fibrillin-1 or fibronectin Arg-Gly-Asp cell-binding fragments, cells showed heparin-dependent spreading and focal contact formation in response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions.     

Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease

Properdin binds to proximal tubular epithelial cells (PTEC) and activates the complement system via the alternative pathway in vitro. Cellular ligands for properdin in the kidney have not yet been identified. Because properdin interacts with solid-phase heparin, we investigated whether heparan sulfate proteoglycans (HSPG) could be the physiological ligands of properdin. Kidneys from proteinuric rats showed colocalization of syndecan-1, a major epithelial HSPG, and properdin in the apical membranes of PTEC, which was not seen in control renal tissue. In vitro, PTEC did not constitutively express properdin. However, exogenous properdin binds to these cells in a dose-dependent fashion. Properdin binding was prevented by heparitinase pretreatment of the cells and was dose-dependently inhibited by exogenous heparin. ELISA and surface plasmon resonance spectroscopy (BIAcore) showed a strong dose-dependent interaction between heparan sulfate (HS) and properdin (K(d) = 128 nm). Pretreatment of HSPG with heparitinase abolished this interaction in ELISA. Competition assays, using a library of HS-like polysaccharides, showed that sulfation pattern, chain length, and backbone composition determine the interaction of properdin with glycosaminoglycans. Interestingly, two nonanticoagulant heparin derivatives inhibited properdin-HS interaction in ELISA and BIAcore. Incubation of PTEC with human serum as complement source led to complement activation and deposition of C3 on the cells. This C3 deposition is dependent on the binding of properdin to HS as shown by heparitinase pretreatment of the cells. Our data identify tubular HS as a novel docking platform for alternative pathway activation via properdin, which might play a role in proteinuric renal damage. Our study also suggests nonanticoagulant heparinoids may provide renoprotection in complement-dependent renal diseases.     

Enzymatic modification of heparan sulfate on a biochip promotes its interaction with antithrombin III

A heparan sulfate glycosaminoglycan chain, biotinylated at its reducing-end, was bound to a streptavidin-coated biochip. Surface plasmon resonance spectroscopy showed a low affinity interaction with antithrombin III (ATIII) when it was flowed over a surface containing heparan sulfate. ATIII bound tightly with high affinity when the same surface was enzymatically modified to using 3-O-sulfotransferase isoform 1 (3-OST-1) in the presence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The 3-OST-1 enzyme is involved in heparan sulfate biosynthesis and introduces a critical 3-O-sulfo group into this glycosaminoglycan affording the appropriate pentasaccharide sequence capable of high affinity binding to ATIII. This experiment demonstrates the specific structural modification of a glycosaminoglycan bound to a biochip using a biosynthetic enzyme, suggesting a new approach to rapid screening glycosaminoglycan-protein interactions.     

Eotaxin selectively binds heparin. An interaction that protects eotaxin from proteolysis and potentiates chemotactic activity in vivo

An important feature of chemokines is their ability to bind to the glycosaminoglycan (GAG) side chains of proteoglycans, predominately heparin and heparan sulfate. To date, all chemokines tested bind to immobilized heparin in vitro, as well as cell surface heparan sulfate in vitro and in vivo. These interactions play an important role in modulating the action of chemokines by facilitating the formation of stable chemokine gradients within the vascular endothelium and directing leukocyte migration, by protecting chemokines from proteolysis, by inducing chemokine oligomerization, and by facilitating transcytosis. Despite the importance of eotaxin in eosinophil differentiation and recruitment being well established, little is known about the interaction between eotaxin and GAGs and the functional consequences of such an interaction. Here we report that eotaxin binds selectively to immobilized heparin with high affinity (K(d) = 1.23 x 10(-8) M), but not to heparan sulfate or a range of other GAGs. The interaction of eotaxin with heparin does not promote eotaxin oligomerization but protects eotaxin from proteolysis directly by plasmin and indirectly by cathepsin G and elastase. In vivo, co-administration of eotaxin and heparin is able to significantly enhance eotaxin-mediated eosinophil recruitment in a mouse air-pouch model. Furthermore, when heparin is co-administered with eotaxin at a concentration that does not normally result in eosinophil infiltration, eosinophil recruitment occurs. In contrast, heparin does not enhance eotaxin-mediated eosinophil chemotaxis in vitro, suggesting protease protection or haptotactic gradient formation as the mechanism by which heparin enhances eotaxin action in vivo. These results suggest a role for mast cell-derived heparin in the recruitment of eosinophils, reinforcing Th2 polarization of inflammatory responses.