Simon J. Clark

Structural basis for complement factor H–linked age-related macular degeneration

Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to ∼50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulators surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance–monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG–FH complex.

His-384 Allotypic Variant of Factor H Associated with Age-related Macular Degeneration Has Different Heparin Binding Properties from the Non-disease-associated Form

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.

Shiga Toxin Activates Complement and Binds Factor H: Evidence for an Active Role of Complement in Hemolytic Uremic Syndrome

Infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of hemolytic uremic syndrome (HUS). Shiga toxins (Stxs), especially Stx2, are believed to represent major virulence factors of EHEC, contributing to HUS pathogenesis. Beside EHEC-associated HUS, there are hereditary atypical forms of HUS, which are mostly caused by mutations of complement regulators. The aim of the present study was to investigate whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors. Purified Stx2 markedly activated complement via the alternative pathway and was found to bind to factor H (FH), however, only when it was active. No apparent cleavage or destruction of FH was visible, and cofactor activity in fluid phase was unaffected, but clearly delayed for surface-attached FH, where it is essential for host cell protection. Binding studies using FH constructs revealed that Stx2 binds to short consensus repeats (SCRs) 6–8 and SCRs18–20, but not to SCRs16–17, i.e., to regions involved in the surface recognition function of FH. In conclusion, complement, and in particular FH, not only plays an important role in atypical HUS, but most probably also in EHEC-induced HUS.

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.

Impaired Binding of the Age-related Macular Degeneration-associated Complement Factor H 402H Allotype to Bruch's Membrane in Human Retina

Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruchs membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruchs membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruchs membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.

Tissue-Specific Host Recognition by Complement Factor H Is Mediated by Differential Activities of Its Glycosaminoglycan-Binding Regions

Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan with a highly diverse range of structures, for which two regions of CFH (CCP6–8 and CCP19–20) have been implicated in HS binding. Mutations/polymorphisms within these glycosaminoglycan-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome. In this study, we demonstrate that CFH has tissue-specific binding properties mediated through its two HS-binding regions. Our data show that the CCP6–8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCP19–20, and that their sulfate specificities are different. Furthermore, the HS binding site in CCP6–8, which is affected by the AMD-associated Y402H polymorphism, plays the principal role in host tissue recognition in the human eye, whereas the CCP19–20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and atypical hemolytic uremic syndrome, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.

Genetic variants in the complement system predisposing to age-related macular degeneration: A review ☆

Age-related macular degeneration (AMD) is a major cause of visual impairment in the western world. It is characterized by the presence of lipoproteinaceous deposits (drusen) in the inner layers of the retina. Immunohistochemistry studies identified deposition of complement proteins in the drusen as well as in the choroid. In the last decade, genetic studies have linked both common and rare variants in genes of the complement system to increased risk of development of AMD. Here, we review the variants described to date and discuss the functional implications of dysregulation of the alternative pathway of complement in AMD.

Complement factor H and age-related macular degeneration: The role of glycosaminoglycan recognition in disease pathology

AMD (age-related macular degeneration) is the major cause of blindness in the western world, associated with the formation of extracellular deposits called drusen in the macula, i.e. the central region of the retina. These drusen contain cellular debris and proteins, including components of the complement system such as the regulator CFH (complement factor H); dysregulation of complement is thought to play a major role in the development of AMD. CFH acts through its capacity to recognize polyanionic structures [e.g. sulfated GAGs (glycosaminoglycans)] found on host tissues, and thereby inactivates any C3b that becomes deposited. Importantly, a common polymorphism in CFH (Y402H) has been strongly associated with an increased risk of AMD. This polymorphism, which causes a tyrosine to histidine coding change, has been shown to alter the binding of CFH to sulfated GAGs, as well as to other ligands including C-reactive protein, necrotic cells and bacterial coat proteins. Of these, the change in the GAG-recognition properties of CFH is likely to be of most significance to AMD. Recent research has revealed that the disease-associated 402H allotype interacts less well (compared with 402Y) with binding sites within the macula (e.g. Bruchs membrane), where the GAGs heparan sulfate and dermatan sulfate play a major role in mediating the interaction with CFH. Reduced binding of the 402H allotype could result in impaired regulation of complement leading to chronic local inflammation that may contribute to the accumulation of drusen and thus the initiation, development and progression of AMD.

Inflammation as a predictor for delayed cerebral ischemia after aneurysmal subarachnoid haemorrhage

Background The mechanism of development of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH) is poorly understood. Inflammatory processes are implicated in the development of ischemic stroke and may also predispose to the development of DCI following SAH. The objective of this study was to test whether concentrations of circulating inflammatory markers (C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin 1 receptor antagonist (IL-1Ra)) were predictive for DCI following SAH. Secondary analyses considered white cell count (WCC) and erythrocyte sedimentation rate (ESR). Methods This was a single-center case-control study nested within a prospective cohort. Plasma inflammatory markers were measured in patients up to 15 days after SAH (initial, peak, average, final and rate of change to final). Cases were defined as those developing DCI. Inflammatory markers were compared between cases and randomly selected matched controls. Results Among the 179 participants there were 46 cases of DCI (26%). In primary analyses the rate of change of IL-6 was associated with DCI (OR 2.3 (95% CI 1.1 to 5.0); p=0.03). The final value and rate of change of WCC were associated with DCI (OR 1.2 (95% CI 1.0 to 1.3) and OR 1.3 (95% CI 1.0 to 1.6), respectively). High values of ESR were associated with DCI (OR 2.4 (95% CI 1.3 to 4.6) initial; OR 2.3 (95% CI 1.3 to 4.2) average; OR 2.1 (95% CI 1.1 to 3.9) peak; and OR 2.0 (95% CI 1.2 to 3.3) final value). Conclusions Leucocytosis and change in IL-6 prior to DCI reflect impending cerebral ischemia. The time-independent association of ESR with DCI after SAH may identify this as a risk factor. These data suggest that systemic inflammatory mechanisms may increase the susceptibility to the development of DCI after SAH.

Mapping the differential distribution of glycosaminoglycans in the adult human retina, choroid and sclera

PURPOSE. To map the distribution of different classes of glycosaminoglycans (GAGs) in the healthy human retina, choroid, and sclera. METHODS. Frozen tissue sections were made from adult human donor eyes. The GAG chains of proteoglycans (PGs) were detected with antibodies directed against various GAG structures (either directly or after pretreatment with GAG-degrading enzymes); hyaluronan (HA) was detected using biotinylated recombinant G1-domain of human versican. The primary detection reagents were identified with FITC-labeled probes and analyzed by fluorescence microscopy. RESULTS. Heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and HA were present throughout the retina and choroid, but keratan sulfate (KS) was detected only in the sclera. HS labeling was particularly strong in basement membrane-containing structures, the nerve fiber layer (NFL), and retinal pigment epithelium (RPE)-for example, intense staining was seen with an antibody that binds strongly to sequences containing 3-O-sulfation in the internal limiting membrane (ILM) and in the basement membrane of blood vessels. Unsulfated CS was seen throughout the retina, particularly in the ILM and interphotoreceptor matrix (IPM) with 6-O-sulfated CS also prominent in the IPM. There was labeling for DS throughout the retina and choroid, especially in the NFL, ganglion cell layer, and blood vessels. CONCLUSIONS. The detection of GAG chains with specific probes and fluorescence microscopy provides for the first time a detailed analysis of their compartmentalization in the human retina, by both GAG chain type and sulfation pattern. This reference map provides a basis for understanding the functional regulation of GAG-binding proteins in health and disease processes.