- Email: [email protected]
Insight into the role of CFHR proteins in renal disease
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Abstracts / Immunobiology 221 (2016) 1131–1225
182 Molecular and functional characterization of guinea pig mannose-binding lectin Yurie Baba 1,∗ , Chiaki Ono 1 , Reo Sekiguchi 2 , Hirotaka Narisawa 1 , Koichiro Tateishi 1 , Yuka Morita 1 , Masaru Nonaka 2 , Misao Matsushita 1 1 Department of Applied Biochemistry, Tokai University, Hiratsuka, Japan 2 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
Mannose-binding lectin (MBL) is a serum protein that plays an important role in innate immunity by activating the lectin complement pathway in association with MBL-associated serine protease (MASP). We report the characterization of guinea pig MBL in terms of its molecular structure and function. To elucidate the primary structure of guinea pig MBL, PCR was first carried out using primers designed based on conserved nucleotide sequences of MBL cDNA from various mammals and guinea pig liver cDNA as a template. After amplification, two PCR products with slightly different sizes were obtained. By cloning of these cDNAs and subsequent RACE (rapid amplification of cDNA ends), the complete amino acid sequences of two homologous isoforms of guinea pig MBL, MBLA and MBL-C, were determined. MBL-A and MBL-C consist of 249 and 245 amino acids respectively, including putative signal peptides and show 52% identity. We also isolated the MBL protein from guinea pig serum by using GlcNAc-agarose and ion exchange chromatography. The molecular size of the purified guinea pig MBL was 32 kDa under reducing conditions on SDS-PAGE. Its N-terminal amino acid sequence was identical to the predicted N-terminal amino acid sequence of MBL-C. Collagenase digestion of the guinea pig MBL preparation yielded a band of 22 kDa. The guinea pig MBL preparation showed the ability to activate the lectin pathway upon binding to GlcNAc, suggesting the presence of MASP in the preparation. In addition, the guinea pig MBL preparation activated the alternative pathway without involvement of C4. http://dx.doi.org/10.1016/j.imbio.2016.06.197 183 Insight into the role of CFHR proteins in renal disease Josué Gutiérrez Tenorio 1,2,∗ , Sheila Pinto García 3 , Jesús García-Fernández 3 , Emilia Arjona 1,2,3 , Santiago Rodriguez de Córdoba 3 , Elena Goicoechea de Jorge 1,2 1
Departamento de Inmunología, Facultad de Medicina, Universidad Complutense, Madrid, Spain 2 Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain 3 Centro de Investigaciones Biológicas and Ciber de Enfermedades Raras, Madrid, Spain Over the past few years, very robust genetic evidence has accumulated supporting a significant role for the complement factor H-related proteins (FHRs) in various diseases including age-related macular degeneration (AMD), atypical haemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G) and, more recently, IgA nephropathy (IgAN). However, and despite specific associations have been established between rare or common polymorphisms in the CFHR genes with increased risk or protection to these diseases, the biological explanation behind these associations is largely unknown. Recently, we described that FHR-1, FHR-2 and FHR-5
circulate in plasma as oligomers (homo- or hetero-oligomers) and demonstrated a role for these proteins as deregulators of the complement system by competing off factor H (FH) for C3b binding. Moreover, we showed that the very peculiar C3G-associated CFHR1, 2 and 5 genetic variants involving duplication of the FHR dimerization domains are gain-of-function mutants that compete better with FH, leading to increased complement activation. Despite these advances, the exact biological role of the FHRs remains obscure and the interpretation of the biological consequences of most CFHR genetic variants found associated with disease is a significant challenge. In order to gain insights into the pathogenic mechanisms by which the CFHRs may lead to renal disease, we performed an in depth retrospective analysis of the genetic variability and plasma proteins levels in a cohort of aHUS (n = 280) and C3G (n = 90) Spanish patients. Here we report the identification of numerous genetic variants in the CFHR1-5 genes in both aHUS and C3G patients. We have also established the normal range of variation of FHR-1, FHR2 and FHR-5 in plasma and determined how those levels differ in the aHUS and C3G populations. Moreover, performing familial studies we determined that the plasma levels of these proteins are influenced by disease activity. Overall, these data illustrate the complexity of the CFHR1-5 locus and of its association with disease, and demonstrate both, common FHR features to aHUS and C3G, as well as FHR peculiarities that seem to be specific of aHUS or C3G. http://dx.doi.org/10.1016/j.imbio.2016.06.198
184 Mannan-binding lectin (MBL) associated serine proteases in mice Simon A. Mortensen, Annette G. Hansen, Esben Axelgaard, Frederik D. Hansen, Steffen Thiel ∗ , Jens C. Jensenius Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus, Denmark E-mail address: [email protected] (S. Thiel). The collectins, mannan-binding lectin and CL-LK activate the complement cascade via activation of the associated serine proteases, MASPs. While the MASPs have been extensively studied in humans their counterparts in mice have received only little attention. We wish to present the results of some early investigations aimed at rectifying this situation. The concentrations of MASP-1, MASP-3, and MAp44 in mouse serum were thus examined. Polyclonal antibodies against murine MASP-1, MASP-3, and MAp44 were raised in rabbits and a Time-Resolved Immuno Fluoro Metric Assay (TRIFMA) was constructed. TRIFMA essentially works as does ELISA, only the enzyme system is exchanged with estimation of the time resolved fluorescence of the europium ion. In our case the developing antibodies are biotinylated and the europium ion is attached to streptavidin. Quantifications of MASP-1, MASP-3, and MAp44 were carried out on a mouse serum pool. The assays were also tested for cross reactivity with other species (goat, sheep, pig, cow, rat, rabbit, chicken, and horse). Cross reactions were seen with goat, sheep, pig, and cow MAp44, with goat, sheep, and pig MASP-3, whereas no cross reactions were seen for MASP-1. The serum levels of MASP-1, MASP-3, and MAp44 in 11 laboratory mice strains were compared and found to vary about a factor of ten. We furthermore investigated the half-life of MASP-1, MASP-3, and MAp44 in masp-1 knockout mice and found that MASP-1 and
Abstracts / Immunobiology 221 (2016) 1131–1225
182 Molecular and functional characterization of guinea pig mannose-binding lectin Yurie Baba 1,∗ , Chiaki Ono 1 , Reo Sekiguchi 2 , Hirotaka Narisawa 1 , Koichiro Tateishi 1 , Yuka Morita 1 , Masaru Nonaka 2 , Misao Matsushita 1 1 Department of Applied Biochemistry, Tokai University, Hiratsuka, Japan 2 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
Mannose-binding lectin (MBL) is a serum protein that plays an important role in innate immunity by activating the lectin complement pathway in association with MBL-associated serine protease (MASP). We report the characterization of guinea pig MBL in terms of its molecular structure and function. To elucidate the primary structure of guinea pig MBL, PCR was first carried out using primers designed based on conserved nucleotide sequences of MBL cDNA from various mammals and guinea pig liver cDNA as a template. After amplification, two PCR products with slightly different sizes were obtained. By cloning of these cDNAs and subsequent RACE (rapid amplification of cDNA ends), the complete amino acid sequences of two homologous isoforms of guinea pig MBL, MBLA and MBL-C, were determined. MBL-A and MBL-C consist of 249 and 245 amino acids respectively, including putative signal peptides and show 52% identity. We also isolated the MBL protein from guinea pig serum by using GlcNAc-agarose and ion exchange chromatography. The molecular size of the purified guinea pig MBL was 32 kDa under reducing conditions on SDS-PAGE. Its N-terminal amino acid sequence was identical to the predicted N-terminal amino acid sequence of MBL-C. Collagenase digestion of the guinea pig MBL preparation yielded a band of 22 kDa. The guinea pig MBL preparation showed the ability to activate the lectin pathway upon binding to GlcNAc, suggesting the presence of MASP in the preparation. In addition, the guinea pig MBL preparation activated the alternative pathway without involvement of C4. http://dx.doi.org/10.1016/j.imbio.2016.06.197 183 Insight into the role of CFHR proteins in renal disease Josué Gutiérrez Tenorio 1,2,∗ , Sheila Pinto García 3 , Jesús García-Fernández 3 , Emilia Arjona 1,2,3 , Santiago Rodriguez de Córdoba 3 , Elena Goicoechea de Jorge 1,2 1
Departamento de Inmunología, Facultad de Medicina, Universidad Complutense, Madrid, Spain 2 Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain 3 Centro de Investigaciones Biológicas and Ciber de Enfermedades Raras, Madrid, Spain Over the past few years, very robust genetic evidence has accumulated supporting a significant role for the complement factor H-related proteins (FHRs) in various diseases including age-related macular degeneration (AMD), atypical haemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G) and, more recently, IgA nephropathy (IgAN). However, and despite specific associations have been established between rare or common polymorphisms in the CFHR genes with increased risk or protection to these diseases, the biological explanation behind these associations is largely unknown. Recently, we described that FHR-1, FHR-2 and FHR-5
circulate in plasma as oligomers (homo- or hetero-oligomers) and demonstrated a role for these proteins as deregulators of the complement system by competing off factor H (FH) for C3b binding. Moreover, we showed that the very peculiar C3G-associated CFHR1, 2 and 5 genetic variants involving duplication of the FHR dimerization domains are gain-of-function mutants that compete better with FH, leading to increased complement activation. Despite these advances, the exact biological role of the FHRs remains obscure and the interpretation of the biological consequences of most CFHR genetic variants found associated with disease is a significant challenge. In order to gain insights into the pathogenic mechanisms by which the CFHRs may lead to renal disease, we performed an in depth retrospective analysis of the genetic variability and plasma proteins levels in a cohort of aHUS (n = 280) and C3G (n = 90) Spanish patients. Here we report the identification of numerous genetic variants in the CFHR1-5 genes in both aHUS and C3G patients. We have also established the normal range of variation of FHR-1, FHR2 and FHR-5 in plasma and determined how those levels differ in the aHUS and C3G populations. Moreover, performing familial studies we determined that the plasma levels of these proteins are influenced by disease activity. Overall, these data illustrate the complexity of the CFHR1-5 locus and of its association with disease, and demonstrate both, common FHR features to aHUS and C3G, as well as FHR peculiarities that seem to be specific of aHUS or C3G. http://dx.doi.org/10.1016/j.imbio.2016.06.198
184 Mannan-binding lectin (MBL) associated serine proteases in mice Simon A. Mortensen, Annette G. Hansen, Esben Axelgaard, Frederik D. Hansen, Steffen Thiel ∗ , Jens C. Jensenius Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus, Denmark E-mail address: [email protected] (S. Thiel). The collectins, mannan-binding lectin and CL-LK activate the complement cascade via activation of the associated serine proteases, MASPs. While the MASPs have been extensively studied in humans their counterparts in mice have received only little attention. We wish to present the results of some early investigations aimed at rectifying this situation. The concentrations of MASP-1, MASP-3, and MAp44 in mouse serum were thus examined. Polyclonal antibodies against murine MASP-1, MASP-3, and MAp44 were raised in rabbits and a Time-Resolved Immuno Fluoro Metric Assay (TRIFMA) was constructed. TRIFMA essentially works as does ELISA, only the enzyme system is exchanged with estimation of the time resolved fluorescence of the europium ion. In our case the developing antibodies are biotinylated and the europium ion is attached to streptavidin. Quantifications of MASP-1, MASP-3, and MAp44 were carried out on a mouse serum pool. The assays were also tested for cross reactivity with other species (goat, sheep, pig, cow, rat, rabbit, chicken, and horse). Cross reactions were seen with goat, sheep, pig, and cow MAp44, with goat, sheep, and pig MASP-3, whereas no cross reactions were seen for MASP-1. The serum levels of MASP-1, MASP-3, and MAp44 in 11 laboratory mice strains were compared and found to vary about a factor of ten. We furthermore investigated the half-life of MASP-1, MASP-3, and MAp44 in masp-1 knockout mice and found that MASP-1 and