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1.
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.  相似文献   

2.
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.  相似文献   

3.
The main virulence factor of group A streptococcus (GAS), M protein, binds plasma complement regulators factor H (FH) and FH-like protein 1 (FHL-1) leading to decreased opsonization. The M protein binding site on FH is within domain 7 in which also the age-related macular degeneration (AMD)-associated polymorphism Y402H is located. We studied if FH allotypes 402H and 402Y have different binding affinities to GAS. Plasma-derived FH allotype 402H and its recombinant fragment FH5-7(402H) showed decreased binding to several GAS strains. Growth of GAS in human blood taken from FH(402H) homozygous individuals was decreased when compared with blood taken from FH(402Y) homozygous individuals. The effect of the allotype 402H can be explained by combining the previous M protein mutagenesis data and the recently published crystal structure of FH6-8. In conclusion the data indicate that the AMD-associated allotype 402H leads to diminished binding of FH to GAS and increased opsonophagocytosis of the bacteria in blood. These results suggest that the homozygous presence of the allele 402H could be associated with decreased risk for severe GAS infections offering an explanation for the high frequency of the allele despite its association with visual impairment.  相似文献   

4.
FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K(d) of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, R(G), of FH in the presence of heparin fragments 18-36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with full-length FH gave K(d) values of 1-3 μM, and similar but weaker K(d) values of 4-20 μM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces.  相似文献   

5.
Factor H (FH) is a major regulator of complement alternative pathway activation. It is composed of 20 short complement regulator (SCR) domains and is genetically associated as a risk factor for age-related macular degeneration. Previous studies on FH suggested that it existed in monomeric and dimeric forms. Improved X-ray scattering and analytical ultracentrifugation methodology for wild-type FH permitted a clarification of these oligomeric properties. Data at lower concentrations revealed a dependence of the X-ray radius of gyration values on concentration that corresponded to the weak self-association of FH. Global sedimentation equilibrium fits indicated that a monomer-dimer equilibrium best described the data up to 1.3 mg/ml with a fitted dissociation constant KD of 28 μM and that higher oligomers formed at increased concentrations. The KD showed that about 85-95% of serum FH will be monomeric in the absence of other factors. Size-distribution analyses in sedimentation velocity experiments showed that monomeric FH was the major species but that as many as six oligomeric forms co-existed with it. The data were explained in terms of two weak dimerisation sites recently identified in the SCR-6/8 and SCR-16/20 fragments of FH with similar KD values. These observations indicate a mechanism for the progressive self-association of FH and may be relevant for complement regulation and the formation of drusen deposits that are associated with age-related macular degeneration.  相似文献   

6.
A single nucleotide polymorphism, tyrosine at position 402 to histidine (Y402H), within the gene encoding complement factor H (FH) predisposes individuals to acquiring age-related macular degeneration (AMD) after aging. This polymorphism occurs in short consensus repeat (SCR) 7 of FH and results in decreased binding affinity of SCR6-8 for heparin. As FH is responsible for regulating the complement system, decreased affinity for heparin results in decreased regulation on surfaces of self. To understand the involvement of the Y402H polymorphism in AMD, we leverage methods from bioinformatics and computational biophysics to quantify structural and dynamical differences between SCR7 isoforms that contribute to decreased pattern recognition in SCR7H402. Our data from molecular and Brownian dynamics simulations suggest a revised mechanism for decreased heparin binding. In this model, transient contacts not observed in structures for SCR7 are predicted to occur in molecular dynamics simulations between coevolved residues Y402 and I412, stabilizing SCR7Y402 in a conformation that promotes association with heparin. H402 in the risk isoform is less likely to form a contact with I412 and samples a larger conformational space than Y402. We observe energy minima for sidechains of Y402 and R404 from SCR7Y402 that are predicted to associate with heparin at a rate constant faster than energy minima for sidechains of H402 and R404 from SCR7H402. As both carbohydrate density and degree of sulfation decrease with age in Bruch’s membrane of the macula, the decreased heparin recognition of SCR7H402 may contribute to the pathogenesis of AMD.  相似文献   

7.
Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.  相似文献   

8.
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 RG 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.  相似文献   

9.
Complement factor H (FH) is an important regulator of the alternative complement pathway. The Y402H polymorphism within the seventh short consensus repeat of FH was recently shown to be associated with age-related macular degeneration, the most common cause of irreversible blindness in the Western world. We examined the effects of this polymorphism on various FH functions. FH purified from sera of age-related macular degeneration patients homozygous for the FH(402H) variant showed a significantly reduced binding to C-reactive protein (CRP), an acute phase protein, as compared with FH derived from unaffected controls homozygous for the FH(402Y) variant. Strongly reduced binding to CRP was also observed with a recombinant fragment of FH (short consensus repeat 5-7) containing the same amino acid change. Because the interaction of CRP and FH promotes complement-mediated clearance of cellular debris in a noninflammatory fashion, we propose that the reduced binding of FH(402H) to CRP could lead to an impaired targeting of FH to cellular debris and a reduction in debris clearance and enhanced inflammation along the macular retinal pigmented epithelium-choroid interface in individuals with age-related macular degeneration.  相似文献   

10.
Factor H (FH) of the complement system acts as a regulatory cofactor for the factor I-mediated cleavage of C3b and binds to polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains. A set of 12 missense mutations in the C-terminal domains between SCR-16 to SCR-20 is associated with haemolytic uraemic syndrome. Recent structural models for intact FH permit the molecular interpretation of these amino acid substitutions. As all nine SCR-20 substitutions correspond to normal amounts of FH in plasma, and were localised in mostly surface-exposed positions, these are inferred to lead to a functional defect in FH. The nine substitutions occur in the same spatial region of SCR-20. As this surface coincides with conserved basic residues in the C-terminal SCR-20 domain, the substitutions provide direct evidence for a polyanionic binding surface. The positions of these conserved basic residues coincide with those of heparin-binding residues in the crystal structure of the acidic fibroblast growth factor-heparin complex. A tenth substitution and another conserved basic residue in SCR-19 are proximate to this binding site. As the remaining FH substitutions could also be correlated with their proximity to conserved basic residues, haemolytic uraemic syndrome may result from a failure of FH to interact with polyanions at cell surfaces in the kidney.  相似文献   

11.
Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina. By scattering, FH at 2.8-7.0 μM was unaffected until [Zn] increased to 20 μM, whereupon the radius of gyration, RG, values increased from 9 to 15 nm at [Zn] = 200 μM. The maximum dimension of FH increased from 32 to 50 nm, indicating that compact oligomers had formed. By ultracentrifugation, size-distribution analyses showed that monomeric FH at 5.57 S was the major species at [Zn] up to 60 μM. At [Zn] above 60 μM, a series of large oligomers were formed, ranging up to 100 S in size. Oligomerisation was reversed by ethylenediaminetetraacetic acid. Structurally distinct large oligomers were observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers and Mg and Ca showed no change. Fluid-phase assays showed reduced FH activities that correlated with increased oligomer formation. The results were attributed to different degrees of stabilisation of weak self-dimerisation sites in FH by transition metals. The relevance of metal-induced FH oligomer formation to complement regulation and age-related macular degeneration is discussed.  相似文献   

12.
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 KD1 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.  相似文献   

13.
Age-related macular degeneration (AMD) is a multifactorial disease that is strongly associated with the Tyr402His variant in the complement factor H (CFH) gene. Drusen are hallmark lesions of AMD and consist of focal-inflammatory and/or immune-mediated depositions of extracellular material at the interface of the retinal pigment epithelium (RPE) and the Bruch membrane. We evaluated the role of CFH in 30 probands with early-onset drusen and identified heterozygous nonsense, missense, and splice variants in five families. The affected individuals all carried the Tyr402His AMD risk variant on the other allele. This supports an autosomal-recessive disease model in which individuals who carry a CFH mutation on one allele and the Tyr402His variant on the other allele develop drusen. Our findings strongly suggest that monogenic inheritance of CFH variants can result in basal laminar drusen in young adults, and this can progress to maculopathy and severe vision loss later in life.  相似文献   

14.
Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.  相似文献   

15.
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 (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His(402) and Tyr(402) variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His(402) and Tyr(402) CCP7 and showed them to be nearly identical. The side chains of His/Tyr(402) have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr(402) CCP7 bound significantly more tightly than His(402) CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr(402) CCP6-8 binds significantly more tightly than His(402) CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.  相似文献   

16.
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.  相似文献   

17.
Age-related macular degeneration is a leading form of blindness in Western countries and is associated with a common SNP (rs 1061170/Y402H) in the Factor H gene, which encodes the two complement inhibitors Factor H and FHL1. However, the functional consequences of this Tyr(402) His exchange in domain 7 are not precisely defined. In this study, we show that the Tyr(402) His sequence variation affects Factor H surface recruitment by monomeric C-reactive protein (mCRP) to specific patches on the surface of necrotic retinal pigment epithelial cells. Enhanced attachment of the protective Tyr(402) variants of both Factor H and FHL1 by mCRP results in more efficient complement control and further provides an anti-inflammatory environment. In addition, we demonstrate that mCRP is generated on the surface of necrotic retinal pigment epithelial cells and that this newly formed mCRP colocalizes with the cell damage marker annexin V. Bound to the cell surface, Factor H-mCRP complexes allow complement inactivation and reduce the release of the proinflammatory cytokine TNF-α. This mCRP-mediated complement inhibitory and anti-inflammatory activity at necrotic membrane lesions is affected by residue 402 of Factor H and defines a new role for mCRP, for Factor H, and also for the mCRP-Factor H complex. The increased protective capacity of the Tyr(402) Factor H variant allows better and more efficient clearance and removal of cellular debris and reduces inflammation and pathology.  相似文献   

18.
The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (Rg) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.  相似文献   

19.
Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.  相似文献   

20.
The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19–20 (FH19–20) and 5–7 (FH5–7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5–7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5–7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5–7. We found that although FH5–7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5–7 domains. Furthermore, binding of FH5–7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5–7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5–7 and regulates alternative pathway activation on plasma HDL particles.  相似文献   

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