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NFY and NFAT transcription factor binding sites are important for expression of the C5a Receptor in the human monocytic U937 cell line
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Abstracts / Molecular Immunology 46 (2009) 2818–2871
O6 3D models of the C3bB and C3bBb complexes: A structural framework to understand regulatory mechanisms and diseaseassociated mutations Agustín Tortajada a,b , Eva Torreira a , Tamara Llorca a , Santiago Rodríguez de Córdoba a,b
Montes a,b , Oscar
a
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain b Centro de Investigación Biomédica en Enfermedades Raras and Instituto Reina Sofía de Investigaciones Nefrológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain C3-convertases are unstable bimolecular complexes that play a critical role in the activation of the complement system. Generation of the alternative pathway (AP) C3-convertase is initiated by the binding of factor B (fB) to its ligand C3b, in a Mg2+ dependent manner, to form the pro-convertase C3bB. Subsequently the serine protease factor D (fD) cleaves fB, releasing the Ba fragment and producing the active complex C3bBb. Recently, we have generated stable complexes of C3bB and C3bBb, using Ni+2 and the convertase-stabilizing mutant fB-279G, respectively, and determined their 3D structure at ∼27 Å resolution using single-particle electron microscopy (EM). These EM models have illustrated key features of the assembly process and the activation of the AP C3convertase (Torreira et al., PNAS 106:882–887, 2009). The structure of C3bB shows that fB undergoes conformational transitions upon binding to C3b, switching from a close to an open conformation in which both Bb and Ba domains interact with C3b and the site of cleavage by fD becomes exposed. We have now extended our observations with the analysis of the 3D structure of the pro-convertase C3bB in the presence of Mg2+ . Together our C3bB and C3bBb 3D models provide a valuable structural framework to understand how complement control proteins regulate the formation and stability of the AP C3-convertase. These models are an important tool to evaluate potential functional consequences of disease-associated mutations and polymorphisms in complement proteins. doi:10.1016/j.molimm.2009.05.192 O7 Putative CCAAT/NFY and NFAT transcription factor binding sites are important for expression of the C5a Receptor in the human monocytic U937 cell line Elizabeth A. Palmer ∗ , Matthew I. Stott, Carmen W. van den Berg Department of Pharmacology, Oncology and Radiology, Cardiff University, School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom C5a exerts its biological actions by binding to the C5a Receptor (C5aR) and expression of C5aR has been found to increase in numerous inflammatory diseases. Despite this, little is known about molecular regulation of expression of the C5aR. To study the regulation of C5aR expression at the transcription level, a 2 kbp fragment upstream from the start codon was cloned into an EGFP-reporter vector. We also investigated the role of a putative AU-rich region in the 3 UTR of the C5aR mRNA in the expression of the C5aR; this C5aR 3 UTR was cloned 3 of the EGFP gene in the EF1a-pEGFP-reporter vector. U937 (human monocytic cell line) cells were stably transfected with these constructs and with various deletion/mutation constructs. Expression of the EGFP-reporter protein was measured using flow-cytometry. The majority of the cloned promoter region (−2037 bp to −201 bp), was found to be dispensable for promoter
activity. Strong regulatory activity was identified between −200 bp and −101 bp as its deletion resulted in a 40% decreased activity compared to the 2 kbp fragment. Site directed mutagenesis of putative CCAAT/NFY (−123 bp to −119 bp) and NFAT (−93 bp to −87 bp) transcription factor binding sites resulted in 60% and 100% decrease in activity compared to the wild type fragment. Deletion of a putative NFB site (−238 bp to −232 bp) had little effect. Expression of the 3 UTR of the C5aR showed reduced expression compared to the plasmid containing the SV40 3 UTR, but site directed mutagenesis of the two AU-rich regions did not affect the expression of the reporter protein suggesting that neither of these AU-rich regions are important for mRNA stability of the C5aR. Our data might be critical for determination of transcription factors that can be targeted pharmacologically to modulate the expression of the C5aR in disease. doi:10.1016/j.molimm.2009.05.193 Session 2: Complement genetics and deficiencies (6th September, 11:10-12:40) O8 Immunodeficiency associated with FCN3 mutation and Ficolin-3 deficiency Tina Hummelshøj ∗ , Christian Honoré, Lea Munthe-Fog, Hans O. Madsen, Henrik Permin, Peter Garred Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark Background: Ficolin-3 (H-ficolin) is expressed in the lung and liver and is one of the recognition molecules in the lectin pathway of complement. Heterozygosity for a FCN3 frame shift mutation (FCN3 + 1637delC, rs28357092) leading to a distortion of the Cterminal end of the molecules has been described among healthy Caucasian individuals with an allele frequency of 0.011. We hypothesised that this variant in the homozygous situation may confer risk for immunodeficiency. Methods: The FCN3 + 1637delC mutation was analysed in 1282 patients with suspected non-HIV related primary immunodeficiency. Ficolin-3 serum concentration and structure were analysed by ELISA, SDS-PAGE and western blotting and complement activation was studied using a Ficolin-3-dependent C4 deposition assay. Results: Twenty-three patients were heterozygous for the variant allele, while one was homozygous. The homozygous FCN3 + 1637delC allele patient suffered from severe unexplained recurrent bacterial lung infections, brochiectasis, lung fibrosis and obstructive lung disease and had experienced an incidence of cerebral abscesses. Median Ficolin-3 serum concentration in the wildtypes was 27.5 g/ml (3.5–42.6) while it was 14.1 g/ml (8.0–31.4 g/l) in heterozygotes (P < 1.0 × 10−8 ). No Ficolin-3 antigen could be detected in the FCN3 + 1637delC homozygous patient, while the oligomeric structure appeared the same in wildtype and in heterozygotes. Complement deposition was absent in Ficolin-3 deficient serum on an acetylated compound, but could be reconstituted with recombinant Ficolin-3.Conclusions: A mutation leading to Ficolin-3 deficiency may cause a novel recessive complement deficiency syndrome associated with severe recurrent lung infections, bronchiectasis, lung fibrosis and obstructive lung disease. doi:10.1016/j.molimm.2009.05.194
Abstracts / Molecular Immunology 46 (2009) 2818–2871
O6 3D models of the C3bB and C3bBb complexes: A structural framework to understand regulatory mechanisms and diseaseassociated mutations Agustín Tortajada a,b , Eva Torreira a , Tamara Llorca a , Santiago Rodríguez de Córdoba a,b
Montes a,b , Oscar
a
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain b Centro de Investigación Biomédica en Enfermedades Raras and Instituto Reina Sofía de Investigaciones Nefrológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain C3-convertases are unstable bimolecular complexes that play a critical role in the activation of the complement system. Generation of the alternative pathway (AP) C3-convertase is initiated by the binding of factor B (fB) to its ligand C3b, in a Mg2+ dependent manner, to form the pro-convertase C3bB. Subsequently the serine protease factor D (fD) cleaves fB, releasing the Ba fragment and producing the active complex C3bBb. Recently, we have generated stable complexes of C3bB and C3bBb, using Ni+2 and the convertase-stabilizing mutant fB-279G, respectively, and determined their 3D structure at ∼27 Å resolution using single-particle electron microscopy (EM). These EM models have illustrated key features of the assembly process and the activation of the AP C3convertase (Torreira et al., PNAS 106:882–887, 2009). The structure of C3bB shows that fB undergoes conformational transitions upon binding to C3b, switching from a close to an open conformation in which both Bb and Ba domains interact with C3b and the site of cleavage by fD becomes exposed. We have now extended our observations with the analysis of the 3D structure of the pro-convertase C3bB in the presence of Mg2+ . Together our C3bB and C3bBb 3D models provide a valuable structural framework to understand how complement control proteins regulate the formation and stability of the AP C3-convertase. These models are an important tool to evaluate potential functional consequences of disease-associated mutations and polymorphisms in complement proteins. doi:10.1016/j.molimm.2009.05.192 O7 Putative CCAAT/NFY and NFAT transcription factor binding sites are important for expression of the C5a Receptor in the human monocytic U937 cell line Elizabeth A. Palmer ∗ , Matthew I. Stott, Carmen W. van den Berg Department of Pharmacology, Oncology and Radiology, Cardiff University, School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom C5a exerts its biological actions by binding to the C5a Receptor (C5aR) and expression of C5aR has been found to increase in numerous inflammatory diseases. Despite this, little is known about molecular regulation of expression of the C5aR. To study the regulation of C5aR expression at the transcription level, a 2 kbp fragment upstream from the start codon was cloned into an EGFP-reporter vector. We also investigated the role of a putative AU-rich region in the 3 UTR of the C5aR mRNA in the expression of the C5aR; this C5aR 3 UTR was cloned 3 of the EGFP gene in the EF1a-pEGFP-reporter vector. U937 (human monocytic cell line) cells were stably transfected with these constructs and with various deletion/mutation constructs. Expression of the EGFP-reporter protein was measured using flow-cytometry. The majority of the cloned promoter region (−2037 bp to −201 bp), was found to be dispensable for promoter
activity. Strong regulatory activity was identified between −200 bp and −101 bp as its deletion resulted in a 40% decreased activity compared to the 2 kbp fragment. Site directed mutagenesis of putative CCAAT/NFY (−123 bp to −119 bp) and NFAT (−93 bp to −87 bp) transcription factor binding sites resulted in 60% and 100% decrease in activity compared to the wild type fragment. Deletion of a putative NFB site (−238 bp to −232 bp) had little effect. Expression of the 3 UTR of the C5aR showed reduced expression compared to the plasmid containing the SV40 3 UTR, but site directed mutagenesis of the two AU-rich regions did not affect the expression of the reporter protein suggesting that neither of these AU-rich regions are important for mRNA stability of the C5aR. Our data might be critical for determination of transcription factors that can be targeted pharmacologically to modulate the expression of the C5aR in disease. doi:10.1016/j.molimm.2009.05.193 Session 2: Complement genetics and deficiencies (6th September, 11:10-12:40) O8 Immunodeficiency associated with FCN3 mutation and Ficolin-3 deficiency Tina Hummelshøj ∗ , Christian Honoré, Lea Munthe-Fog, Hans O. Madsen, Henrik Permin, Peter Garred Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark Background: Ficolin-3 (H-ficolin) is expressed in the lung and liver and is one of the recognition molecules in the lectin pathway of complement. Heterozygosity for a FCN3 frame shift mutation (FCN3 + 1637delC, rs28357092) leading to a distortion of the Cterminal end of the molecules has been described among healthy Caucasian individuals with an allele frequency of 0.011. We hypothesised that this variant in the homozygous situation may confer risk for immunodeficiency. Methods: The FCN3 + 1637delC mutation was analysed in 1282 patients with suspected non-HIV related primary immunodeficiency. Ficolin-3 serum concentration and structure were analysed by ELISA, SDS-PAGE and western blotting and complement activation was studied using a Ficolin-3-dependent C4 deposition assay. Results: Twenty-three patients were heterozygous for the variant allele, while one was homozygous. The homozygous FCN3 + 1637delC allele patient suffered from severe unexplained recurrent bacterial lung infections, brochiectasis, lung fibrosis and obstructive lung disease and had experienced an incidence of cerebral abscesses. Median Ficolin-3 serum concentration in the wildtypes was 27.5 g/ml (3.5–42.6) while it was 14.1 g/ml (8.0–31.4 g/l) in heterozygotes (P < 1.0 × 10−8 ). No Ficolin-3 antigen could be detected in the FCN3 + 1637delC homozygous patient, while the oligomeric structure appeared the same in wildtype and in heterozygotes. Complement deposition was absent in Ficolin-3 deficient serum on an acetylated compound, but could be reconstituted with recombinant Ficolin-3.Conclusions: A mutation leading to Ficolin-3 deficiency may cause a novel recessive complement deficiency syndrome associated with severe recurrent lung infections, bronchiectasis, lung fibrosis and obstructive lung disease. doi:10.1016/j.molimm.2009.05.194