Structure shows that a glycosaminoglycan and protein recognition site in factor H is perturbed by age-related macular degeneration-linked single nucleotide polymorphism

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.     

Basal laminar drusen caused by compound heterozygous variants in the CFH gene

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.     

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.     

Cigarette smoking strongly modifies the association of complement factor H variant and the risk of lung cancer

The complement system is an important immunosurveillance mechanism against tumors, and complement factor H (CFH) is a key regulator for activation of the complement system. Expression of complement factor H has been demonstrated in cell lines from several malignancies. In this study we examined the contribution of the single-nucleotide polymorphism (SNP) Try402His (Y402H) (rs1061170) in the CFH gene to the risk of lung cancer in a case-control study with 1000 cases and 1000 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed by logistic regression. The frequencies for CFH Y402H genotypes among the cases were statistically significantly different from those among controls (χ(2)=8.66, P=0.003), with 402His/His or 402His/Try genotypes being over-represented among patients compared with controls (13.6% versus 9.4%, P<0.004). A multivariate regression analysis showed that a significantly increased risk of lung cancer for the 402His/His or 402His/Try carriers with OR (95% CI), 1.50 (1.12-2.00). When stratified by smoking status, the elevated risk of the cancer associated with variant CFH genotypes was observed among smokers, but not among non-smokers. When analyzed with cumulative smoking dose (pack-years), a super-multiplicative interaction was observed at different smoking levels. Among carriers with the 402Tyr/His or His/His genotype, the ORs of developing lung cancer for smoking<16, 16-28, or >28 pack-years were 0.98 (0.49-1.94), 2.36 (1.14-4.90), and 6.39 (3.49-11.68), respectively. These findings suggest that CFH Y402H polymorphism may interact with cigarette smoking to effect the development of lung cancer in the Chinese population.     

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.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

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.     

Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4^−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4^−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4^−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.     

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.     

The factor H variant associated with age-related macular degeneration (His-384) and the non-disease-associated form bind differentially to C-reactive protein, fibromodulin, DNA, and necrotic cells

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.     

Altered function of factor I caused by amyloid beta: implication for pathogenesis of age-related macular degeneration from Drusen

The results of recent studies have implicated local inflammation and complement activation as the processes involved in the pathogenesis of age-related macular degeneration (AMD). We have demonstrated that amyloid beta (Abeta), which is deposited in drusen, causes an imbalance in the angiogenesis-related factors in retinal pigment epithelial cells. We have also shown that neprilysin gene-disrupted mice accumulate Abeta, and develop several features of AMD. The purpose of this study was to investigate the mechanisms involved in the development of AMD that are triggered by Abeta. Our results showed that Abeta binds to complement factor I which inhibits the ability of factor I to cleave C3b to inactivated iC3b. Factor H and factor I are soluble complement-activation inhibitors, and preincubation of factor I with Abeta in the presence of factor H abolished the ability of Abeta to cleave C3b, and also abolished the ability of factor I to cleave FGR-AMC. In contrast, Abeta did not affect the function of factor H even after binding. The production of iC3b was significantly decreased when C3b and factor H were incubated with the eyes from neprilysin gene-disrupted mice as compared with when C3b and factor H were incubated with eyes from age-matched wild-type mice. These results suggest that Abeta activates the complement system within drusen by blocking the function of factor I leading to a low-grade, chronic inflammation in subretinal tissues. These findings link four factors that have been suggested to be associated with AMD: inflammation, complement activation, Abeta deposition, and drusen.     

Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.     

Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack

Metastatic cancer cells, like trophoblasts of the developing placenta, are invasive and must escape immune surveillance to survive. Complement has long been thought to play a significant role in the tumor surveillance mechanism. Bone sialoprotein (BSP) and osteopontin (OPN, ETA-1) are expressed by trophoblasts and are strongly up-regulated by many tumors. Indeed, BSP has been shown to be a positive indicator of the invasive potential of some tumors. In this report, we show that BSP and OPN form rapid and tight complexes with complement Factor H. Besides its key role in regulating complement-mediated cell lysis, Factor H also appears to play a role when "hijacked" by invading organisms in enabling cellular evasion of complement. We have investigated whether BSP and OPN may play a similar role in tumor cell complement evasion by testing to see whether these glycoproteins could promote tumor cell survival. Recombinant OPN and BSP can protect murine erythroleukemia cells from attack by human complement as well as human MCF-7 breast cancer cells and U-266 myeloma cells from attack by guinea pig complement. The mechanism of this gain of function by tumor cell expression of BSP or OPN has been defined using specific peptides and antibodies to block BSP and OPN protective activity. The expression of BSP and OPN in tumor cells provides a selective advantage for survival via initial binding to alpha(V)beta(3) integrin (both) or CD44 (OPN) on the cell surface, followed by sequestration of Factor H to the cell surface and inhibition of complement-mediated cell lysis.     

Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations

Factor H is a regulatory glycoprotein of the complement system. We expressed the three N-terminal complement control protein modules of human factor H (FH1-3) and confirmed FH1-3 to be the minimal unit with cofactor activity for C3b proteolysis by factor I. We reconstructed FH1-3 from NMR-derived structures of FH1-2 and FH2-3 revealing an approximately 105-A-long rod-like arrangement of the modules. In structural comparisons with other C3b-engaging proteins, factor H module 3 most closely resembles factor B module 3, consistent with factor H competing with factor B for binding C3b. Factor H modules 1, 2, and 3 each has a similar backbone structure to first, second, and third modules, respectively, of functional sites in decay accelerating factor and complement receptor type 1; the equivalent intermodular tilt and twist angles are also broadly similar. Resemblance between molecular surfaces is closest for first modules but absent in the case of second modules. Substitution of buried Val-62 with Ile (a factor H single nucleotide polymorphism potentially protective for age-related macular degeneration and dense deposit disease) causes rearrangements within the module 1 core and increases thermal stability but does not disturb the interface with module 2. Replacement of partially exposed (in module 1) Arg-53 by His (an atypical hemolytic uremic syndrome-linked mutation) did not impair structural integrity at 37 degrees C, but this FH1-2 mutant was less stable at higher temperatures; furthermore, chemical shift differences indicated potential for small structural changes at the module 1-2 interface.     

Vitamin A receptors. II. Characteristics of retinol binding in chick retina and pigment epithelium

Gel filtration studies demonstrate that retinol receptors of chick retinal and pigment epithelial cytosols are (1) of very similar nature (2) of small molecular size (about 18000 daltons) and are different in character from serum proteins. Citral inhibits the binding of [3H]retinol to the retinal 2 S receptor. Retinol acetate competes with retinol for binding to 2 S receptor in both retina and pigment epithelium whereas retinol palmitate is an effective competitor only in the pigment epithelium. Dithiothreitol maximizes 2 S binding in retina and pigment epithelial cytosol; its absence does not lead to receptor aggregation however. A limited number of high affinity binding sites (2 S receptor) appear to be present in retina and pigment epithelium. A 5 S binding species is also present in pigment epithelium; it is similar in character to [3H]retinol binding in serum and may arise from serum contamination of the pigment epithelial preparation. Binding affinity in retina is high with possibly two classes of retinol binding sites present of KD about 1 - 10(-9) and 4 - 10(-8).     

Dysregulation in retinal para-inflammation and age-related retinal degeneration in CCL2 or CCR2 deficient mice

We have shown previously that a para-inflammatory response exists at the retinal/choroidal interface in the aging eye; and this response plays an important role in maintaining retinal homeostasis under chronic stress conditions. We hypothesized that dysregulation of the para-inflammatory response may result in an overt pro-inflammatory response inducing retinal degeneration. In this study, we examined this hypothesis in mice deficient in chemokine CCL2 or its cognate receptor CCR2. CCL2- or CCR2-deficient mice developed retinal degenerative changes with age, characterized as retinal pigment epithelial (RPE) cell and photoreceptor cell death. Retinal cell death was associated with significantly more subretinal microglial accumulation and increased complement activation. In addition, monocytes from CCL2- or CCR2-deficient mice had reduced capacity for phagocytosis and chemotaxis, expressed less IL-10 but more iNOS, IL-12 and TNF-α when compared to monocytes from WT mice. Complement activation at the site of RPE cell death resulted in C3b/C3d but not C5b-9 deposition, indicating only partial activation of the complement pathway. Our results suggest that altered monocyte functions may convert the protective para-inflammatory response into an overtly harmful inflammation at the retina/choroidal interface in CCL2- or CCR2-deficient mice, leading to RPE and photoreceptor degeneration. These data support a concept whereby a protective para-inflammatory response relies upon a normally functioning innate immune system. If the innate immune system is deficient chronic stress may tip the balance towards an overt inflammatory response causing cell/tissue damage.     

Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein

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.     

Complement factor H deficiency combined with smoking promotes retinal degeneration in a novel mouse model

Age-related macular degeneration is the leading cause of blindness in the elderly. The Y402H polymorphism in complement factor H promotes disease-like pathogenesis, and a Cfh^+/− murine model can replicate this phenotype, but only after two years. We reasoned that by combining CFH deficiency with cigarette smoke exposure, we might be able to accelerate disease progression to facilitate preclinical research in this disease. Wild-type and Cfh^+/− mice were exposed to nose-only cigarette smoke for three months. Retinal tissue morphology and visual function were evaluated by optical coherence tomography, fundus photography and autofluorescence, and electroretinogram. Retinal pigment epithelial cell phenotype and ultrastructure were evaluated by immunofluorescence staining and transmission electron microscopy. Cfh^+/− smoking mice showed a dome-like protruding lesion at the ellipsoid zone (drusen-like deposition), many retinal hyper-autofluorescence spots, and a marked decrease in A- and B-wave amplitudes. Compared with non-smoking mice, wild-type and Cfh^+/− smoking mice showed sub-retinal pigment epithelium complement protein 3 deposition, activation of microglia, metabolic waste accumulation, and impairment of tight junctions. Microglia cells migrated into the photoreceptor outer segment layer in Cfh^+/− smoking mice showed increased activation. Our results suggest that exposing Cfh^+/− mice to smoking leads to earlier onset of age-related macular degeneration than in other animal models, which may facilitate preclinical research into the pathophysiology and treatment of this disease.     

Complement dysregulation in AMD: RPE-Bruch's membrane-choroid

The question as to why the macula of the retina is prone to an aging disease (age-related macular degeneration) remains unanswered. This unmet challenge has implications since AMD accounts for approximately 54% of blindness in the USA (Swaroop, Chew, Bowes Rickman and Abecasis, 2009). While AMD has onset in the elder years, it likely develops over time. Genetic discovery to date has accounted for approximately 50% of the inheritable component of AMD. The polymorphism that has been most widely studied is the Y402H allele in the complement factor H gene. The implication of this genetic association is that in a subset of AMD cases, unregulated complement activation is permissive for AMD. Given that this gene variant results in an amino acid substitution, it is assumed that this change will have functional consequences although the precise mechanisms are still unknown. Genetic predisposition is not the only factor however, since in this complex disease there is substantial evidence that lifestyle factors such as diet and smoking contribute to risk. Here we provide an overview of current knowledge with respect to factors involved in AMD pathogenesis. Interwoven with these issues is a discussion of the significant role played by aging processes, some of which are unique to the retina and retinal pigment epithelium. One recurring theme is the potential for disease promotion by diverse types of oxidation products.     

Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.     

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β₂-glycoprotein I and interferes with innate immune recognition.