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Structure-Function-Disease map for the genetic abnormalities in the central complement component C3
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Abstracts / Molecular Immunology 48 (2011) 1666–1733
O25 Human complement factor H-related protein 2 (CFHR2) represents a novel complement regulator, which is reduced in a patient with MPGN I H.E. Eberhardt ∗ , Q. Chen, P. Zipfel, C. Skerka Hans-Knöll Institute, Jena, Germany Introduction: Membranoproliferative glumerulonephritis (MPGN) is a rare kidney disease that manifests with proteinuria, hematuria and acute nephrotic syndrome. The pathology of MPGN is caused by dysregulation of the alternative pathway of complement activation, strong activation of C3 and subendothelial deposition of immunglobulins with C3 or deposits of C3 cleavage products along the glomerular basement membrane. Here we describe a heterozygous deletion of the complement factor H related protein 2 (CFHR2) in a patient with MPGN type I, which leads to severely decreased CFHR2 serum levels. The physiological function of the CFHR2 protein is still unknown which let us investigate the role of this protein in complement regulation. Methods: MLPA, ELISA, Western blot, flow cytometry, and hemolytic assay. Results: Here we identify the CFHR2 protein as a regulator of the alternative pathway C3-convertase and of the terminal complex formation (TCC). CFHR2 and CFHR2 deletion mutants were recombinantly expressed in the yeast expression system Pichia pastoris. Binding studies revealed interaction of CFHR2 with the central complement component C3b, and the binding region was located to the C-terminal SCRs 3–4. CFHR2 inhibited C3-convertase activity as indicated by reduced C3a generation in complement activation assays with NHS. In addition CFHR2 also inhibited terminal complex formation which was mediated by SCRs1-2. In addition CFHR2 bound to necrotic HUVEC cells and limited complement activation as shown by reduced iC3b levels. Conclusion: These data describe CFHR2, a novel human complement regulator. Absence or reduction of complement control by CFHR2 are likely linked to the development of MPGN. doi:10.1016/j.molimm.2011.06.245 O26 Crystal structure of MASP-2 in complex with a highly selective phage display evolved inhibitor V. Harmat a,∗ , I. Héja b , K. Menyhárd c , J. Dobó d , P. Závodszky d , P. Gál d , G. Pál b a
Eötvös Loránd University-Hungarian Academy of Sciences, Budapest, Hungary b Eötvös Loránd University, Department of Biochemistry, Budapest, Hungary c Eötvös Loránd University, Institute of Chemistry, Budapest, Hungary d Hungarian Academy of Sciences, Institute of Enzymology, Budapest, Hungary Introduction: Activator enzymes of the classical and lectin pathways of the complement are serine proteases, which have exquisite substrate selectivity, yet possess an overlapping set of natural substrates. Substrate binding sites of C1s, MASP-1 and MASP-2 are quite different. Moreover, each enzyme has substrates with differing sequences around the cleavage site. The above facts suggest that in addition to the canonical enzyme/substrate interactions extended interactions and/or flexibility contribute to substrate recognition of these enzymes. Methods: We developed via in vitro evolution techniques highly selective nanomolar inhibitors of MASP-1 and MASP-2 based on
pacifastin trypsin inhibitor scaffold. We co-crystallized the catalytic fragment of MASP-2 with the highest affinity MASP-2 inhibitor clone having the consensus evolved sequence to explore the structural basis of the interactions. The structure was solved and refined to 1.9 resolution. Dynamic aspects of inhibitor binding of MASPs were characterized by molecular dynamics simulations. Results: The structure reveals that both the inhibitor and MASP2 underwent significant conformational changes. The evolved sequence that ensures high selectivity is accommodated by 10 H-bonds including S1-P1 salt bridge and several hydrophobic contacts. However the contact region involving more than 50% of the inhibitor residues and several MASP-2 loops extends over the classical serine protease/inhibitor binding sites significantly. Conclusions: Besides explaining high selectivity and inhibitory activity, the MASP-2/inhibitor structure also demonstrates shape adaptivity of MASP-2. Its comparison with uncomplexed MASP-2 and with MASP-2/MASP-2 enzyme/product complex reveals that loop plasticity of MASP-2 allows for accommodating different protein targets with extended yet partially different interaction regions. doi:10.1016/j.molimm.2011.06.246 O27 Structure-Function-Disease map for the genetic abnormalities in the central complement component C3 L.T. Roumenina a,∗ , L. Halbwachs-Mecarelli b , P. Bordereau a , T. Rybkine a , M.A. Dragon-Durey c , M. Noris d , C. Sautes-Fridman a , V. Frémeaux-Bacchi c a
INSERM UMRS 872, Paris, France INSERM U845, Hôpital Necker, Paris, France c AP-HP Hôpital Européen Georges-Pompidou, Paris, France d Mario Negri Institute for Pharmacological Research, Ranica, Italy b
Introduction: Genetic abnormalities in complement proteins are related to number of diseases. Complete C3 deficiencies are known to predispose to infections. Recently heterozygous C3 mutations have been reported in two kidney diseases—atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy. The objective of this study is to analyse the structure-function relationship for a large number of C3 mutations. Methods: 32 mutations in C3 gene were analysed in this study. All C3 mutations were mapped on the structures of C3, C3b, C3bFH1-4, C3d-FH19-20, C3b-FB and C3bBb. Results: C3 mutations are associated with low or normal C3 levels in patients’ plasma. Using transient transfection, both type I (quantitative deficiency) and type II (normal level of the mutant protein) mutations were identified. Among the type II mutations, 75% appeared to be clustered in one particular surface and co-localize with the binding sites for Factor H CCP1–4, CCP19–20, MCP and Factor B. 10% are located in the newly identified substrate binding site of the C3 convertase. 15% are far from any known binding site. Ninety-two percent of these mutations were found in aHUS patients and 8%—in C3 glomerulopathy. Functional analysis demonstrated perturbation of the function for the majority of the tested mutations. All complete C3 deficiencies were due to stop-codons and conferred susceptibly to infections. Surprisingly, type I mutations were found in aHUS. Conclusion: Disease-associated C3 mutations cluster in functionally important regions identified by recent crystal structures. Disease-specific segregation of the mutation was observed. Functional studies are in progress. doi:10.1016/j.molimm.2011.06.247
Abstracts / Molecular Immunology 48 (2011) 1666–1733
O25 Human complement factor H-related protein 2 (CFHR2) represents a novel complement regulator, which is reduced in a patient with MPGN I H.E. Eberhardt ∗ , Q. Chen, P. Zipfel, C. Skerka Hans-Knöll Institute, Jena, Germany Introduction: Membranoproliferative glumerulonephritis (MPGN) is a rare kidney disease that manifests with proteinuria, hematuria and acute nephrotic syndrome. The pathology of MPGN is caused by dysregulation of the alternative pathway of complement activation, strong activation of C3 and subendothelial deposition of immunglobulins with C3 or deposits of C3 cleavage products along the glomerular basement membrane. Here we describe a heterozygous deletion of the complement factor H related protein 2 (CFHR2) in a patient with MPGN type I, which leads to severely decreased CFHR2 serum levels. The physiological function of the CFHR2 protein is still unknown which let us investigate the role of this protein in complement regulation. Methods: MLPA, ELISA, Western blot, flow cytometry, and hemolytic assay. Results: Here we identify the CFHR2 protein as a regulator of the alternative pathway C3-convertase and of the terminal complex formation (TCC). CFHR2 and CFHR2 deletion mutants were recombinantly expressed in the yeast expression system Pichia pastoris. Binding studies revealed interaction of CFHR2 with the central complement component C3b, and the binding region was located to the C-terminal SCRs 3–4. CFHR2 inhibited C3-convertase activity as indicated by reduced C3a generation in complement activation assays with NHS. In addition CFHR2 also inhibited terminal complex formation which was mediated by SCRs1-2. In addition CFHR2 bound to necrotic HUVEC cells and limited complement activation as shown by reduced iC3b levels. Conclusion: These data describe CFHR2, a novel human complement regulator. Absence or reduction of complement control by CFHR2 are likely linked to the development of MPGN. doi:10.1016/j.molimm.2011.06.245 O26 Crystal structure of MASP-2 in complex with a highly selective phage display evolved inhibitor V. Harmat a,∗ , I. Héja b , K. Menyhárd c , J. Dobó d , P. Závodszky d , P. Gál d , G. Pál b a
Eötvös Loránd University-Hungarian Academy of Sciences, Budapest, Hungary b Eötvös Loránd University, Department of Biochemistry, Budapest, Hungary c Eötvös Loránd University, Institute of Chemistry, Budapest, Hungary d Hungarian Academy of Sciences, Institute of Enzymology, Budapest, Hungary Introduction: Activator enzymes of the classical and lectin pathways of the complement are serine proteases, which have exquisite substrate selectivity, yet possess an overlapping set of natural substrates. Substrate binding sites of C1s, MASP-1 and MASP-2 are quite different. Moreover, each enzyme has substrates with differing sequences around the cleavage site. The above facts suggest that in addition to the canonical enzyme/substrate interactions extended interactions and/or flexibility contribute to substrate recognition of these enzymes. Methods: We developed via in vitro evolution techniques highly selective nanomolar inhibitors of MASP-1 and MASP-2 based on
pacifastin trypsin inhibitor scaffold. We co-crystallized the catalytic fragment of MASP-2 with the highest affinity MASP-2 inhibitor clone having the consensus evolved sequence to explore the structural basis of the interactions. The structure was solved and refined to 1.9 resolution. Dynamic aspects of inhibitor binding of MASPs were characterized by molecular dynamics simulations. Results: The structure reveals that both the inhibitor and MASP2 underwent significant conformational changes. The evolved sequence that ensures high selectivity is accommodated by 10 H-bonds including S1-P1 salt bridge and several hydrophobic contacts. However the contact region involving more than 50% of the inhibitor residues and several MASP-2 loops extends over the classical serine protease/inhibitor binding sites significantly. Conclusions: Besides explaining high selectivity and inhibitory activity, the MASP-2/inhibitor structure also demonstrates shape adaptivity of MASP-2. Its comparison with uncomplexed MASP-2 and with MASP-2/MASP-2 enzyme/product complex reveals that loop plasticity of MASP-2 allows for accommodating different protein targets with extended yet partially different interaction regions. doi:10.1016/j.molimm.2011.06.246 O27 Structure-Function-Disease map for the genetic abnormalities in the central complement component C3 L.T. Roumenina a,∗ , L. Halbwachs-Mecarelli b , P. Bordereau a , T. Rybkine a , M.A. Dragon-Durey c , M. Noris d , C. Sautes-Fridman a , V. Frémeaux-Bacchi c a
INSERM UMRS 872, Paris, France INSERM U845, Hôpital Necker, Paris, France c AP-HP Hôpital Européen Georges-Pompidou, Paris, France d Mario Negri Institute for Pharmacological Research, Ranica, Italy b
Introduction: Genetic abnormalities in complement proteins are related to number of diseases. Complete C3 deficiencies are known to predispose to infections. Recently heterozygous C3 mutations have been reported in two kidney diseases—atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy. The objective of this study is to analyse the structure-function relationship for a large number of C3 mutations. Methods: 32 mutations in C3 gene were analysed in this study. All C3 mutations were mapped on the structures of C3, C3b, C3bFH1-4, C3d-FH19-20, C3b-FB and C3bBb. Results: C3 mutations are associated with low or normal C3 levels in patients’ plasma. Using transient transfection, both type I (quantitative deficiency) and type II (normal level of the mutant protein) mutations were identified. Among the type II mutations, 75% appeared to be clustered in one particular surface and co-localize with the binding sites for Factor H CCP1–4, CCP19–20, MCP and Factor B. 10% are located in the newly identified substrate binding site of the C3 convertase. 15% are far from any known binding site. Ninety-two percent of these mutations were found in aHUS patients and 8%—in C3 glomerulopathy. Functional analysis demonstrated perturbation of the function for the majority of the tested mutations. All complete C3 deficiencies were due to stop-codons and conferred susceptibly to infections. Surprisingly, type I mutations were found in aHUS. Conclusion: Disease-associated C3 mutations cluster in functionally important regions identified by recent crystal structures. Disease-specific segregation of the mutation was observed. Functional studies are in progress. doi:10.1016/j.molimm.2011.06.247