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1.
Invest Ophthalmol Vis Sci ; 63(12): 30, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36445700

RESUMO

Purpose: Factor H (FH, encoded by CFH) prevents activation of the complement system's alternative pathway (AP) on host tissues. FH impedes C3 convertase (C3bBb) formation, accelerates C3bBb decay, and is a cofactor for factor I (FI)-catalyzed C3b cleavage. Numerous CFH variants are associated with age-related macular degeneration (AMD), but their functional consequences frequently remain undetermined. Here, we conduct functional comparisons between a control version of FH (not AMD linked) and 21 AMD-linked FH variants. Methods: Recombinantly produced, untagged, full-length FH versions were assayed for binding to C3b and decay acceleration of C3bBb using surface-plasmon resonance, FI-cofactor activity using a fluorescent probe of C3b integrity, suppression of C5b-9 assembly on an AP-activating surface, and inhibition of human AP-mediated lysis of sheep erythrocytes. Results: All versions were successfully purified despite below-average yields for Arg2Thr, Arg53Cys, Arg175Pro, Arg175Gln, Ile221Val, Tyr402His, Pro503Ala, Arg567Gly, Gly1194Asp, and Arg1210Cys. Compared to control FH, Arg2Thr, Leu3Val, Ser58Ala, Asp90Gly, Asp130Asn, Gln400Lys, Tyr402His, Gly650Val, Ser890Ile, and Thr965Met showed minimal functional differences. Arg1210C, Arg53His, Arg175Gln, Gly1194Asp, Pro503Ala, Arg53Cys, Arg576Gly, and Arg175Pro (in order of decreasing efficacy) underperformed, while Ile221Val, Arg303Gln, and Arg303Trp were "marginal." We newly identified variants toward the center of the molecule, Pro503Ala and Arg567Gly, as potentially pathogenic. Conclusions: Our approach could be extended to other variants of uncertain significance and to assays for noncanonical FH activities, aiming to facilitate selection of cohorts most likely to benefit from therapeutic FH. This is timely as recombinant therapeutic FH is in development for intravitreal treatment of AMD in patients with reduced FH functionality.


Assuntos
Fator H do Complemento , Degeneração Macular , Animais , Humanos , Aceleração , Fator H do Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento , Proteínas do Sistema Complemento , Degeneração Macular/genética , Ovinos
2.
Curr Eye Res ; 47(7): 1087-1093, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35282732

RESUMO

PURPOSE: GEM103 is a recombinantly produced full-length version of the human complement factor H (CFH) under clinical investigation for treatment of age-related macular degeneration (AMD) in individuals carrying an AMD risk-associated genetic variant of CFH. This study aimed to investigate the complement pathway-related functions of GEM103 in comparison with those of native human CFH. METHODS: Key biological activities of GEM103 and human serum-derived CFH (sdCFH) were compared using four independent functional assays. Assays of C3b binding and C3 convertase decay-accelerating activity (DAA) were performed by surface plasmon resonance (SPR). Cofactor activity (CA) was measured using 8-anilinonaphthalene-1-sulfonic acid as a fluorescent probe of C3b integrity. The abilities of GEM103 and sdCFH to protect sheep erythrocytes from hemolysis by CFH-depleted normal human serum were assessed colorimetrically. RESULTS: In multiple SPR-based assays of C3b binding and DAA, the performance of GEM103 was consistently comparable to that of sdCFH across a range of matching concentrations. The EC50 ± SD in the fluorescence-based fluid-phase CA assay was 0.21 ± 0.06 µM for GEM103 compared to 0.20 ± 0.09 µM for sdCFH. In hemolysis assays, the EC50 value of 0.33 ± 0.16 µM for GEM103 versus 0.46 ± 0.06 µM for sdCFH were not significantly different (p = 0.81). CONCLUSIONS: GEM103, a recombinant CFH developed by Gemini Therapeutics, shows activity profiles comparable to sdCFH in all complement-related assays employed in this study, suggesting that GEM103 is equivalent to the native glycoprotein in terms of its in vitro functional activity. These results support further study of GEM103 as a potential therapy for AMD.


Assuntos
Fator H do Complemento , Degeneração Macular , Animais , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Hemólise , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Polimorfismo de Nucleotídeo Único , Ovinos
3.
Front Immunol ; 12: 681098, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054871

RESUMO

Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen.


Assuntos
Complemento C3/imunologia , Complemento C3/metabolismo , Fator H do Complemento/biossíntese , Fator H do Complemento/deficiência , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Expressão Gênica , Imunomodulação , Camundongos , Camundongos Knockout , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Leveduras/genética , Leveduras/metabolismo
4.
J Biol Chem ; 294(52): 20148-20163, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31719147

RESUMO

Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·µm-2 AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.


Assuntos
Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Microscopia de Força Atômica , Ressonância de Plasmônio de Superfície , Ativação do Complemento , Complemento C3b/química , Complemento C3d/química , Complemento C3d/metabolismo , Fator H do Complemento/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Ligação Proteica
5.
J Biol Chem ; 292(32): 13345-13360, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28637873

RESUMO

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of ES PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Ativação do Complemento , Degeneração Macular/genética , Mutação , Substituição de Aminoácidos , Animais , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , C3 Convertase da Via Alternativa do Complemento/química , C3 Convertase da Via Alternativa do Complemento/genética , C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3d/química , Complemento C3d/genética , Complemento C3d/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Fator I do Complemento/química , Fator I do Complemento/genética , Fator I do Complemento/metabolismo , Eritrócitos/química , Hemólise , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Degeneração Macular/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Carneiro Doméstico , Solubilidade , Streptococcus pneumoniae/metabolismo , Propriedades de Superfície
6.
Biochem Soc Trans ; 43(5): 812-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26517887

RESUMO

Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a ß-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.


Assuntos
Enzimas Ativadoras do Complemento/química , Ativação do Complemento , Proteínas Inativadoras do Complemento/química , Desenho de Fármacos , Modelos Moleculares , Engenharia de Proteínas , Animais , Sítios de Ligação , Enzimas Ativadoras do Complemento/genética , Enzimas Ativadoras do Complemento/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Inativadores do Complemento/química , Inativadores do Complemento/metabolismo , Inativadores do Complemento/farmacologia , Proteínas Inativadoras do Complemento/genética , Proteínas Inativadoras do Complemento/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia
7.
J Immunol ; 195(10): 4986-98, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26459349

RESUMO

In an attempt to evade annihilation by the vertebrate complement system, many microbes capture factor H (FH), the key soluble complement-regulating protein in human plasma. However, FH is normally an active complement suppressor exclusively on self-surfaces and this selective action of FH is pivotal to self versus non-self discrimination by the complement system. We investigated whether the bacterially captured FH becomes functionally enhanced and, if so, how this is achieved at a structural level. We found, using site-directed and truncation mutagenesis, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and cross-linking and mass spectrometry, that the N-terminal domain of Streptococcus pneumoniae protein PspC (PspCN) not only binds FH extraordinarily tightly but also holds it in a previously uncharacterized conformation. Functional enhancement arises from exposure of a C-terminal cryptic second binding site in FH for C3b, the activation-specific fragment of the pivotal complement component, C3. This conformational change of FH doubles its affinity for C3b and increases 5-fold its ability to accelerate decay of the binary enzyme (C3bBb) responsible for converting C3 to C3b in an amplification loop. Despite not sharing critical FH-binding residues, PspCNs from D39 and Tigr4 S. pneumoniae exhibit similar FH-anchoring and enhancing properties. We propose that these bacterial proteins mimic molecular markers of self-surfaces, providing a compelling hypothesis for how FH prevents complement-mediated injury to host tissue while lacking efficacy on virtually all other surfaces. In hemolysis assays with 2-aminoethylisothiouronium bromide-treated erythrocytes that recapitulate paroxysmal nocturnal hemoglobinuria, PspCN enhanced protection of cells by FH, suggesting a new paradigm for therapeutic complement suppression.


Assuntos
Proteínas de Bactérias/química , Complemento C3b/química , Fator H do Complemento/química , Streptococcus pneumoniae/química , Proteínas de Bactérias/imunologia , Complemento C3b/imunologia , Fator H do Complemento/imunologia , Hemoglobinúria Paroxística/imunologia , Humanos , Estrutura Terciária de Proteína , Streptococcus pneumoniae/imunologia
8.
Biochemistry ; 52(23): 3949-62, 2013 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-23701234

RESUMO

Factor H (FH) is a soluble regulator of the proteolytic cascade at the core of the evolutionarily ancient vertebrate complement system. Although FH consists of a single chain of similar protein modules, it has a demanding job description. Its chief role is to prevent complement-mediated injury to healthy host cells and tissues. This entails recognition of molecular patterns on host surfaces combined with control of one of nature's most dangerous examples of a positive-feedback loop. In this way, FH modulates, where and when needed, an amplification process that otherwise exponentially escalates the production of the pro-inflammatory, pro-phagocytic, and pro-cytolytic cleavage products of complement proteins C3 and C5. Mutations and single-nucleotide polymorphisms in the FH gene and autoantibodies against FH predispose individuals to diseases, including age-related macular degeneration, dense-deposit disease, and atypical hemolytic uremic syndrome. Moreover, deletions or variations of genes for FH-related proteins also influence the risk of disease. Numerous pathogens hijack FH and use it for self-defense. As reviewed herein, a molecular understanding of FH function is emerging. While its functional oligomeric status remains uncertain, progress has been achieved in characterizing its three-dimensional architecture and, to a lesser extent, its intermodular flexibility. Models are proposed, based on the reconciliation of older data with a wealth of recent evidence, in which a latent circulating form of FH is activated by its principal target, C3b tethered to a self-surface. Such models suggest hypotheses linking sequence variations to pathophysiology, but improved, more quantitative, functional assays and rigorous data analysis are required to test these ideas.


Assuntos
Ativação do Complemento , Fator H do Complemento/fisiologia , Motivos de Aminoácidos , Sítios de Ligação , Complemento C3b/química , Complemento C3b/fisiologia , Fator H do Complemento/química , Humanos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
9.
J Mol Biol ; 424(5): 295-312, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23017427

RESUMO

The 155-kDa plasma glycoprotein factor H (FH), which consists of 20 complement control protein (CCP) modules, protects self-tissue but not foreign organisms from damage by the complement cascade. Protection is achieved by selective engagement of FH, via CCPs 1-4, CCPs 6-8 and CCPs 19-20, with polyanion-rich host surfaces that bear covalently attached, activation-specific, fragments of complement component C3. The role of intervening CCPs 9-18 in this process is obscured by lack of structural knowledge. We have concatenated new high-resolution solution structures of overlapping recombinant CCP pairs, 10-11 and 11-12, to form a three-dimensional structure of CCPs 10-12 and validated it by small-angle X-ray scattering of the recombinant triple-module fragment. Superimposing CCP 12 of this 10-12 structure with CCP 12 from the previously solved CCP 12-13 structure yielded an S-shaped structure for CCPs 10-13 in which modules are tilted by 80-110° with respect to immediate neighbors, but the bend between CCPs 10 and 11 is counter to the arc traced by CCPs 11-13. Including this four-CCP structure in interpretation of scattering data for the longer recombinant segments, CCPs 10-15 and 8-15, implied flexible attachment of CCPs 8 and 9 to CCP 10 but compact and intimate arrangements of CCP 14 with CCPs 12, 13 and 15. Taken together with difficulties in recombinant production of module pairs 13-14 and 14-15, the aberrant structure of CCP 13 and the variability of 13-14 linker sequences among orthologues, a structural dependency of CCP 14 on its neighbors is suggested; this has implications for the FH mechanism.


Assuntos
Fator H do Complemento/química , Fator H do Complemento/metabolismo , Espectroscopia de Ressonância Magnética , Humanos , Modelos Moleculares , Conformação Proteica
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