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FHL-1 Y402H polymorphism associated with age-related macular degeneration affects multiple functions
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Abstracts / Molecular Immunology 44 (2007) 147–266
interacts with a network of proteins that regulate cell polarity in epithelial cells, neurons and T cells. We investigated the effect of ligation of the immunoregulatory cell surface receptor, CD46, on lymphocyte polarity during antigen presentation and cytotoxic effector function. Ligation of CD46 on human T cells prevented recruitment of the microtubule organizing center (MTOC), CD3 and perforin to the interface with the antigen presenting cell (APC), and caused a reduction in IFN-␥ production. In human NK cells, similar changes in polarity induced by CD46 ligation inhibited the recruitment of the MTOC and perforin to the interface with target cells and correlated with reduced killing. These data indicate that external signals mediated by CD46 can alter lymphocyte polarization towards APC or target cells, inhibiting lymphocyte function. doi:10.1016/j.molimm.2006.07.178 174 The factor H/FHL-1 Y402H polymorphism associated with age-related macular degeneration affects multiple functions Rebecca J. Ormsby a , Josephine Hoh b , Kim M. Griggs a , Tania A. Sadlon a , David L. Gordon a a
Department of Microbiology and Infectious Diseases, Flinders Medical Centre and Flinders University, Bedford Park, SA 5042, Australia; b Department of Epidemiology and Public Health, Yale University, School of Medicine, New Haven, CT, USA Age-related macular degeneration (AMD) is the major cause of irreversible blindness in Western countries affecting over 50 million elderly people worldwide. Recently, several reports have independently identified a Tyr to His polymorphism in the gene encoding factor H (FH) and factor H Like-1 (FHL-1) that is associated with an increased susceptibility for AMD. Factor H is composed of twenty short consensus repeats (SCR), while FHL-1, which is a splice variant of FH, consists of the first seven N-terminal SCR domains of FH. The polymorphism is located in SCR 7 (Y402H), which we have shown to contain the binding site for heparin, C-reactive protein (CRP) and Group A Streptococcal M protein. Using the expression system Pichia pastoris, we have cloned and expressed two recombinant SCR 1–7 constructs containing either the Tyr or His residue at position 402. In addition, we have purified the wildtype and polymorphic variant of FH from sera. Analyses show that there is no effect of the polymorphism on FH fluid-phase cofactor activity or C3b binding when compared to wildtype FH. In contrast, the polymorphism exhibits reduced binding to CRP and M protein. Using heparin affinity chromatography we found that although the polymorphism does not influence the interaction of fulllength FH with heparin, the recombinant SCR 1–7 construct containing the polymorphism has a decreased affinity for heparin. These results indicate that this single amino acid change alters the binding properties of FH/FHL-1 to several ligands and provides a possible pathophysiological explanation for the association of the polymorphism with AMD. doi:10.1016/j.molimm.2006.07.179
175 Localisation of the third and fourth C3b-binding domains in the human complement regulator factor H Rebecca J. Ormsby a , Kim M. Griggs a , David L. Gordon a , T. Sakari Jokiranta b a
Department of Microbiology and Infectious Diseases, Flinders Medical Centre and Flinders University, Bedford Park, SA 5042, Australia; b Department of Bacteriology and Immunology, Haartman Institute and HUSLAB, University and University Hospital of Helsinki, Helsinki, Finland Binding of complement factor H (FH) to C3b facilitates control of the alternative pathway (AP) of complement activation in both plasma and on host cell surfaces and is therefore a critical interaction in AP regulation. Factor H is composed of twenty short consensus repeats (SCRs), and previously three C3b-binding domains have been identified within SCRs 1–4, SCRs 19–20, and in the middle region of FH. To identify the domain(s) in the middle region of FH that interact with C3b, an extensive range of recombinant FH fragments were cloned and expressed in Pichia pastoris. Using a combination of ELISA and surface plasmon resonance analyses we have localised the third C3b-binding domain to SCR 9 and have identified a fourth C3b-binding site within SCRs 12–14. Both C3b-binding domains interact with the C3c region of C3b and compete for binding to C3b. We examined the interaction of SCR 9 with C3b in the presence of heparin and found that heparin does not inhibit the interaction, indicating that the C3b- and heparin-binding sites in SCR 9 are distinct. The utilization of this region by a number of pathogenic bacteria to inhibit complement activation and the recent identification of mutations in the middle region of FH associated with atypical haemolytic uremic syndrome, suggests that this region may play a role in AP regulation. The identification and localisation of functional domains within the mid-region of FH is an important step towards understanding the function of this region in AP regulation. doi:10.1016/j.molimm.2006.07.180 176 Complement component C6 deficiency in the Western Cape, South Africa; segregation of four genetic defects A. Orren a,c , K. Parham a , A.G. Roberts a , A.D. Thomas a , F. Leisegang b , B.P. Morgan a , P.C. Potter c a
Department Medical Biochemistry & Immunology, Cardiff University, Cardiff, UK; b Division Chemical Pathology, University of Cape Town, Cape Town, South Africa; c Allergology Unit, University of Cape Town, Cape Town, South Africa Total deficiency of complement component C6 (C6Q0), although uncommon in the Western Cape, is considerably more frequent than in most other areas where the disease has been studied. Previously, three defects were identified in the Cape, two (1195delC and 1936delG) had already been found independently in African Americans and one is an exon 6 defect which
Abstracts / Molecular Immunology 44 (2007) 147–266
interacts with a network of proteins that regulate cell polarity in epithelial cells, neurons and T cells. We investigated the effect of ligation of the immunoregulatory cell surface receptor, CD46, on lymphocyte polarity during antigen presentation and cytotoxic effector function. Ligation of CD46 on human T cells prevented recruitment of the microtubule organizing center (MTOC), CD3 and perforin to the interface with the antigen presenting cell (APC), and caused a reduction in IFN-␥ production. In human NK cells, similar changes in polarity induced by CD46 ligation inhibited the recruitment of the MTOC and perforin to the interface with target cells and correlated with reduced killing. These data indicate that external signals mediated by CD46 can alter lymphocyte polarization towards APC or target cells, inhibiting lymphocyte function. doi:10.1016/j.molimm.2006.07.178 174 The factor H/FHL-1 Y402H polymorphism associated with age-related macular degeneration affects multiple functions Rebecca J. Ormsby a , Josephine Hoh b , Kim M. Griggs a , Tania A. Sadlon a , David L. Gordon a a
Department of Microbiology and Infectious Diseases, Flinders Medical Centre and Flinders University, Bedford Park, SA 5042, Australia; b Department of Epidemiology and Public Health, Yale University, School of Medicine, New Haven, CT, USA Age-related macular degeneration (AMD) is the major cause of irreversible blindness in Western countries affecting over 50 million elderly people worldwide. Recently, several reports have independently identified a Tyr to His polymorphism in the gene encoding factor H (FH) and factor H Like-1 (FHL-1) that is associated with an increased susceptibility for AMD. Factor H is composed of twenty short consensus repeats (SCR), while FHL-1, which is a splice variant of FH, consists of the first seven N-terminal SCR domains of FH. The polymorphism is located in SCR 7 (Y402H), which we have shown to contain the binding site for heparin, C-reactive protein (CRP) and Group A Streptococcal M protein. Using the expression system Pichia pastoris, we have cloned and expressed two recombinant SCR 1–7 constructs containing either the Tyr or His residue at position 402. In addition, we have purified the wildtype and polymorphic variant of FH from sera. Analyses show that there is no effect of the polymorphism on FH fluid-phase cofactor activity or C3b binding when compared to wildtype FH. In contrast, the polymorphism exhibits reduced binding to CRP and M protein. Using heparin affinity chromatography we found that although the polymorphism does not influence the interaction of fulllength FH with heparin, the recombinant SCR 1–7 construct containing the polymorphism has a decreased affinity for heparin. These results indicate that this single amino acid change alters the binding properties of FH/FHL-1 to several ligands and provides a possible pathophysiological explanation for the association of the polymorphism with AMD. doi:10.1016/j.molimm.2006.07.179
175 Localisation of the third and fourth C3b-binding domains in the human complement regulator factor H Rebecca J. Ormsby a , Kim M. Griggs a , David L. Gordon a , T. Sakari Jokiranta b a
Department of Microbiology and Infectious Diseases, Flinders Medical Centre and Flinders University, Bedford Park, SA 5042, Australia; b Department of Bacteriology and Immunology, Haartman Institute and HUSLAB, University and University Hospital of Helsinki, Helsinki, Finland Binding of complement factor H (FH) to C3b facilitates control of the alternative pathway (AP) of complement activation in both plasma and on host cell surfaces and is therefore a critical interaction in AP regulation. Factor H is composed of twenty short consensus repeats (SCRs), and previously three C3b-binding domains have been identified within SCRs 1–4, SCRs 19–20, and in the middle region of FH. To identify the domain(s) in the middle region of FH that interact with C3b, an extensive range of recombinant FH fragments were cloned and expressed in Pichia pastoris. Using a combination of ELISA and surface plasmon resonance analyses we have localised the third C3b-binding domain to SCR 9 and have identified a fourth C3b-binding site within SCRs 12–14. Both C3b-binding domains interact with the C3c region of C3b and compete for binding to C3b. We examined the interaction of SCR 9 with C3b in the presence of heparin and found that heparin does not inhibit the interaction, indicating that the C3b- and heparin-binding sites in SCR 9 are distinct. The utilization of this region by a number of pathogenic bacteria to inhibit complement activation and the recent identification of mutations in the middle region of FH associated with atypical haemolytic uremic syndrome, suggests that this region may play a role in AP regulation. The identification and localisation of functional domains within the mid-region of FH is an important step towards understanding the function of this region in AP regulation. doi:10.1016/j.molimm.2006.07.180 176 Complement component C6 deficiency in the Western Cape, South Africa; segregation of four genetic defects A. Orren a,c , K. Parham a , A.G. Roberts a , A.D. Thomas a , F. Leisegang b , B.P. Morgan a , P.C. Potter c a
Department Medical Biochemistry & Immunology, Cardiff University, Cardiff, UK; b Division Chemical Pathology, University of Cape Town, Cape Town, South Africa; c Allergology Unit, University of Cape Town, Cape Town, South Africa Total deficiency of complement component C6 (C6Q0), although uncommon in the Western Cape, is considerably more frequent than in most other areas where the disease has been studied. Previously, three defects were identified in the Cape, two (1195delC and 1936delG) had already been found independently in African Americans and one is an exon 6 defect which