Measurement of Factor H Variants in Plasma Using Variant-Specific Monoclonal Antibodies: Application to Assessing Risk of Age-Related Macular Degeneration

Hakobyan, Svetlana; Harris, Claire L.; Tortajada, Agustín; Jorge, Elena Goicoechea de; García‐Layana, Alfredo; Fernández-Robredo, Patricia; Córdoba, Santiago Rodrı́guez de; Morgan, B. Paul

doi:10.1167/iovs.07-1523

Cited by 82 publications

(90 citation statements)

References 50 publications

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“…The FH/FHR3 hybrid protein was purified from patient serum with affinity chromatography using immobilized mAb to FH CCP7 402Y (MBI6) 31 on a 1-ml HiTrap NHS HP Column (GE Healthcare). After washing with PBS, the bound proteins were eluted using PBS/diethylamine (50 mM).…”

Section: Purification Of Fh Speciesmentioning

confidence: 99%

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H–Related 3 Gene in Atypical Hemolytic Uremic Syndrome

Challis¹,

Araujo²,

Wong³

et al. 2015

JASN

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The regulators of complement activation cluster at chromosome 1q32 contains the complement factor H (CFH) and five complement factor H-related (CFHR) genes. This area of the genome arose from several large genomic duplications, and these low-copy repeats can cause genome instability in this region. Genomic disorders affecting these genes have been described in atypical hemolytic uremic syndrome, arising commonly through nonallelic homologous recombination. We describe a novel CFH/CFHR3 hybrid gene secondary to a de novo 6.3-kb deletion that arose through microhomology-mediated end joining rather than nonallelic homologous recombination. We confirmed a transcript from this hybrid gene and showed a secreted protein product that lacks the recognition domain of factor H and exhibits impaired cell surface complement regulation. The fact that the formation of this hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of the genome in which additional genomic disorders may arise.

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Section: Purification Of Fh Speciesmentioning

confidence: 99%

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H–Related 3 Gene in Atypical Hemolytic Uremic Syndrome

Challis¹,

Araujo²,

Wong³

et al. 2015

JASN

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“…FH is a 150-kDa glycoprotein with a plasma concentration ∼250 mg/ ml (9). It regulates the alternative pathway C3 convertase (C3bBb) through three mechanisms.…”

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confidence: 99%

An Engineered Construct Combining Complement Regulatory and Surface-Recognition Domains Represents a Minimal-Size Functional Factor H

Hebecker

Alba-Domínguez

Roumenina

et al. 2013

The Journal of Immunology

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Complement is an essential humoral component of innate immunity; however, its inappropriate activation leads to pathology. Polymorphisms, mutations, and autoantibodies affecting factor H (FH), a major regulator of the alternative complement pathway, are associated with various diseases, including age-related macular degeneration, atypical hemolytic uremic syndrome, and C3 glomerulopathies. Restoring FH function could be a treatment option for such pathologies. In this article, we report on an engineered FH construct that directly combines the two major functional regions of FH: the N-terminal complement regulatory domains and the C-terminal surface-recognition domains. This minimal-size FH (mini-FH) binds C3b and has complement regulatory functions similar to those of the full-length protein. In addition, we demonstrate that mini-FH binds to the FH ligands C-reactive protein, pentraxin 3, and malondialdehyde epitopes. Mini-FH was functionally active when bound to the extracellular matrix and endothelial cells in vitro, and it inhibited C3 deposition on the cells. Furthermore, mini-FH efficiently inhibited complement-mediated lysis of host-like cells caused by a disease-associated FH mutation or by anti-FH autoantibodies. Therefore, mini-FH could potentially be used as a complement inhibitor targeting host surfaces, as well as to replace compromised FH in diseases associated with FH dysfunction.

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“…Plasma or serum C3 and C4 levels were measured using standard nephelometric assays (Siemens). fH, fI, and fB levels were measured by sandwich ELISA as previously described (31)(32)(33). Anti-fH and C3Nef autoantibodies were detected as described previously (34,35).…”

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confidence: 99%

Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

Martı́nez-Barricarte¹,

Heurich²,

et al. 2010

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Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C3 923ΔDG , which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H 2 O) by spontaneous thioester hydrolysis, C3 923ΔDG generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C3b 923ΔDG and C3(H 2 O) 923ΔDG were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase-restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments. IntroductionComplement is a major component of innate immunity, with crucial roles in microbial killing, apoptotic cell clearance, immune complex handling, and modulation of adaptive immune responses. Complement is activated by 3 independent activation pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The critical steps in complement activation are the formation of unstable protease complexes, named complement factor 3-convertases (C3-convertases; specifically, C3bBb for AP and C4b2a for CP and LP), and the cleavage of C3 by the convertases to generate C3b. Convertase-generated C3b can form more AP C3-convertase, providing exponential amplification to the initial activation. Binding of C3b to the C3-convertases generates the C5-convertases with the capacity to bind and cleave C5, initiating formation of the lytic membrane attack complex (MAC). In contrast to the CP and the LP, whose activation is triggered by immune complexes and bacterial mannose groups, respectively, the AP is intrinsically activated. Spontaneous activation of C3 in plasma occurs through the tick-over mecha-

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Measurement of Factor H Variants in Plasma Using Variant-Specific Monoclonal Antibodies: Application to Assessing Risk of Age-Related Macular Degeneration

Abstract: The novel monoclonal antibody MBI-7 was used to develop a robust assay for measurement of fH and the variants in plasma. The simplicity of the assay means that it may be used by any clinical laboratory to identify polymorphic status and to quantify plasma levels in persons at risk for AMD.

Cited by 82 publications

References 50 publications

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H–Related 3 Gene in Atypical Hemolytic Uremic Syndrome

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H–Related 3 Gene in Atypical Hemolytic Uremic Syndrome

An Engineered Construct Combining Complement Regulatory and Surface-Recognition Domains Represents a Minimal-Size Functional Factor H

Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

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