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Carp properdin: Structural and functional diversity of two isotypes
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Abstracts / Immunobiology 221 (2016) 1131–1225
also found that Tecrem expressed on T cells modulates mitogeninduced T cell proliferation in ginbuna crucian carp, indicating that modulation of adaptive immunity is one of the evolutionarily conserved functions of the CD46-like complement regulator. In the present study, we have explored a homeostatic role of Tecrem in the maintenance of fish epithelium, by analyzing expression behavior of Tecrem on an epithelial cell line (KF-1) derived from carp fin. Flow cytometric analysis of Tecrem expression on KF-1 using anti-carp Tecrem monoclonal antibody (MAb) (1F12) suggested that Tecrem expression may be affected by cell aggregation and adhesion. Fluorescent microscopic observation and ELISA-based assay for Tecrem also indicated a role of Tecrem in the adhesion of KF-1 to the surface of culture media. Furthermore, 1F12 MAb deposited on the culture plate significantly enhanced an early stage of cell adhesion process of KF-1. Towards further functional analyses of carp Tecrem, we have prepared recombinant Tecrem proteins in the bacterial expression system. Among the four short consensus repeat (SCR) modules making up the extracellular domains of Tecrem, the N-terminal two SCRs (rSCR1-2) and the C-terminal two SCRs (rSCR3-4) were separately expressed as 6xHis-tagged soluble protein using pColdI vector and Origami B strain. Interaction of the two recombinant domains with carp C3 isotypes and their inhibition of carp complement activation cascades are currently analyzed in parallel with raising polyclonal antibodies, for the clarification of functional modules and their mode of action. http://dx.doi.org/10.1016/j.imbio.2016.06.187 173 Carp properdin: Structural and functional diversity of two isotypes Kazuki Yoshioka ∗ , Yoko Kato-Unoki, Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan Properdin (Pf) is a positive regulator for the activation of the alternative complement pathway (ACP). Pf has also been suggested to be a possible pattern-recognition molecule to trigger ACP activation. Teleost complement system has a striking feature that some of its components are diversified into multiple isoforms with different functions. However this diversity is less characterized for teleost Pf, especially at the protein level. The present study was aimed at elucidating isotypic diversity and functional differentiation of Pf in the common carp (Cyprinus carpio). Molecular cloning of carp Pf revealed two distinct full-length cDNA sequences, CaPf1 and CaPf2, that predict mature proteins composed of seven thrombospondin type 1 modules (TSP0-TSP6), sharing 77% amino acid sequence identity. Genomic Southern hybridization suggested that CaPf1 and CaPf2 are encoded by single each gene in carp genome. Real-time quantitative PCR indicated that expression level of CaPf1 is most abundant in the spleen, whereas CaPf2 was detected mainly in head kidney and renal kidney. Rabbit antisera were raised against their recombinant proteins corresponding to TSP4-6 domains of CaPf1 and CaPf2. Western blotting using anti-CaPf1 and anti-CaPf2 suggested that CaPf1 and CaPf2 are mainly present as a hexamer of polypeptide with molecular weights of 49,000 and 48,000, respectively, in carp serum. Interestingly, CaPf1 and CaPf2 showed different spectra of binding to various microbes after incubation with carp serum, suggesting their functional diversity. EDTA inhibited the binding of either isoform.
CaPf1 and CaPf2 proteins were purified from carp serum by a 5-step procedure composed of precipitation with 5% polyethylenglycol and chromatography on Q-Sepharose HP, Superdex 200, TSKgel SP-5PW, and TSKgel heparin-5PW columns. Interestingly, binding of the purified CaPf1 and CaPf2 to several microbial targets was not detected, in contrast to the recent report on recombinant TSP modules of zebrafish Pf. http://dx.doi.org/10.1016/j.imbio.2016.06.188 174 Up-regulation of C3 levels in cerebrospinal fluid of neuropsychiatric systemic lupus erythematosus patients Tomoyuki Asano 1,∗ , Shuzo Sato 1 , Hiroko Kobayashi 1 , Yoshinobu Kariya 2 , Hiromi Ito 2 , Kyoka Hoshi 2 , Akioh Yoshihara 3 , Yoshikazu Ugawa 3 , Hideharu Sekine 4 , Shunsei Hirohata 5 , Hiroshi Watanabe 1 , Hiromasa Ohira 1 , Yasuhiro Hashimoto 2 1 Department of Gastroenterology and Rheumatology, Fukushima Medical University, Fukushima, Japan 2 Department of Biochemistry, Fukushima Medical University, Fukushima, Japan 3 Department of Neurology, Fukushima Medical University, Fukushima, Japan 4 Department of Immunology, Fukushima Medical University, Fukushima, Japan 5 Department of Rheumatology and Infectious Diseases, Kitasato University, Kanagawa, Japan
Background: Neuropsychiatric manifestation has been reported 40–70% of systemic lupus erythematosus (SLE) patients; it is called neuropsychiatric SLE (NPSLE). Although NPSLE is one of the major causes of death in SLE patients, its pathogenic mechanisms remain to be elucidated. Various autoantibodies have been identified in cerebrospinal fluids (CSF) of NPSLE patients. They are supposed to form immune complexes with complements to induce inflammatory responses. We therefore measured the 3rd component of complement (C3) and other inflammation-related molecules in CSF of NPSLE patients. Methods: This study included 51 SLE patients. According to the 1999 American College of Rheumatology definition criteria, 43 patients were diagnosed with NPSLE whereas 8 patients were diagnosed with non-NPSLE. We measured levels of C3 and IL-6 in CSF and serum by ELISA. Results: C3 levels in CSF of NPSLE (3.68 ± 3.79 g/ml) were significantly higher than that of non-NPSLE (1.75 ± 1.14 g/ml) (p = 0.0071). No significant difference in serum C3 levels was observed between NPSLE (634 ± 311 g/ml) and non-NPLSE (897 ± 374 g/ml) (p = 0.2767). Correlation of C3 levels between CSF and serum was weak in NPSLE (r = 0.2885, p = 0.0380), suggesting that up-regulation of C3 in CSF is independent of that in serum. On the other hand, IL-6 levels in CSF of NPSLE (13.4 ± 1233 pg/ml) were significantly higher than that of nonNPSLE (2.14 ± 2.58 pg/ml) (p = 0.0010), which is consistent with the previous observation report about lupus psychosis. It was notable that levels of C3 and IL-6 in CSF showed strong correlation (r = 0.5102, p = 0.0003). This result suggests that C3 and IL-6 in CSF are up-regulated as a result of series of inflammation. Conclusion: We demonstrated the increase of C3 and IL-6 in CSF of NPSLE patients. One possible explanation for the elevation of C3 may be hyper-production of C3 in the brain. Namely, hyper-
Abstracts / Immunobiology 221 (2016) 1131–1225
also found that Tecrem expressed on T cells modulates mitogeninduced T cell proliferation in ginbuna crucian carp, indicating that modulation of adaptive immunity is one of the evolutionarily conserved functions of the CD46-like complement regulator. In the present study, we have explored a homeostatic role of Tecrem in the maintenance of fish epithelium, by analyzing expression behavior of Tecrem on an epithelial cell line (KF-1) derived from carp fin. Flow cytometric analysis of Tecrem expression on KF-1 using anti-carp Tecrem monoclonal antibody (MAb) (1F12) suggested that Tecrem expression may be affected by cell aggregation and adhesion. Fluorescent microscopic observation and ELISA-based assay for Tecrem also indicated a role of Tecrem in the adhesion of KF-1 to the surface of culture media. Furthermore, 1F12 MAb deposited on the culture plate significantly enhanced an early stage of cell adhesion process of KF-1. Towards further functional analyses of carp Tecrem, we have prepared recombinant Tecrem proteins in the bacterial expression system. Among the four short consensus repeat (SCR) modules making up the extracellular domains of Tecrem, the N-terminal two SCRs (rSCR1-2) and the C-terminal two SCRs (rSCR3-4) were separately expressed as 6xHis-tagged soluble protein using pColdI vector and Origami B strain. Interaction of the two recombinant domains with carp C3 isotypes and their inhibition of carp complement activation cascades are currently analyzed in parallel with raising polyclonal antibodies, for the clarification of functional modules and their mode of action. http://dx.doi.org/10.1016/j.imbio.2016.06.187 173 Carp properdin: Structural and functional diversity of two isotypes Kazuki Yoshioka ∗ , Yoko Kato-Unoki, Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan Properdin (Pf) is a positive regulator for the activation of the alternative complement pathway (ACP). Pf has also been suggested to be a possible pattern-recognition molecule to trigger ACP activation. Teleost complement system has a striking feature that some of its components are diversified into multiple isoforms with different functions. However this diversity is less characterized for teleost Pf, especially at the protein level. The present study was aimed at elucidating isotypic diversity and functional differentiation of Pf in the common carp (Cyprinus carpio). Molecular cloning of carp Pf revealed two distinct full-length cDNA sequences, CaPf1 and CaPf2, that predict mature proteins composed of seven thrombospondin type 1 modules (TSP0-TSP6), sharing 77% amino acid sequence identity. Genomic Southern hybridization suggested that CaPf1 and CaPf2 are encoded by single each gene in carp genome. Real-time quantitative PCR indicated that expression level of CaPf1 is most abundant in the spleen, whereas CaPf2 was detected mainly in head kidney and renal kidney. Rabbit antisera were raised against their recombinant proteins corresponding to TSP4-6 domains of CaPf1 and CaPf2. Western blotting using anti-CaPf1 and anti-CaPf2 suggested that CaPf1 and CaPf2 are mainly present as a hexamer of polypeptide with molecular weights of 49,000 and 48,000, respectively, in carp serum. Interestingly, CaPf1 and CaPf2 showed different spectra of binding to various microbes after incubation with carp serum, suggesting their functional diversity. EDTA inhibited the binding of either isoform.
CaPf1 and CaPf2 proteins were purified from carp serum by a 5-step procedure composed of precipitation with 5% polyethylenglycol and chromatography on Q-Sepharose HP, Superdex 200, TSKgel SP-5PW, and TSKgel heparin-5PW columns. Interestingly, binding of the purified CaPf1 and CaPf2 to several microbial targets was not detected, in contrast to the recent report on recombinant TSP modules of zebrafish Pf. http://dx.doi.org/10.1016/j.imbio.2016.06.188 174 Up-regulation of C3 levels in cerebrospinal fluid of neuropsychiatric systemic lupus erythematosus patients Tomoyuki Asano 1,∗ , Shuzo Sato 1 , Hiroko Kobayashi 1 , Yoshinobu Kariya 2 , Hiromi Ito 2 , Kyoka Hoshi 2 , Akioh Yoshihara 3 , Yoshikazu Ugawa 3 , Hideharu Sekine 4 , Shunsei Hirohata 5 , Hiroshi Watanabe 1 , Hiromasa Ohira 1 , Yasuhiro Hashimoto 2 1 Department of Gastroenterology and Rheumatology, Fukushima Medical University, Fukushima, Japan 2 Department of Biochemistry, Fukushima Medical University, Fukushima, Japan 3 Department of Neurology, Fukushima Medical University, Fukushima, Japan 4 Department of Immunology, Fukushima Medical University, Fukushima, Japan 5 Department of Rheumatology and Infectious Diseases, Kitasato University, Kanagawa, Japan
Background: Neuropsychiatric manifestation has been reported 40–70% of systemic lupus erythematosus (SLE) patients; it is called neuropsychiatric SLE (NPSLE). Although NPSLE is one of the major causes of death in SLE patients, its pathogenic mechanisms remain to be elucidated. Various autoantibodies have been identified in cerebrospinal fluids (CSF) of NPSLE patients. They are supposed to form immune complexes with complements to induce inflammatory responses. We therefore measured the 3rd component of complement (C3) and other inflammation-related molecules in CSF of NPSLE patients. Methods: This study included 51 SLE patients. According to the 1999 American College of Rheumatology definition criteria, 43 patients were diagnosed with NPSLE whereas 8 patients were diagnosed with non-NPSLE. We measured levels of C3 and IL-6 in CSF and serum by ELISA. Results: C3 levels in CSF of NPSLE (3.68 ± 3.79 g/ml) were significantly higher than that of non-NPSLE (1.75 ± 1.14 g/ml) (p = 0.0071). No significant difference in serum C3 levels was observed between NPSLE (634 ± 311 g/ml) and non-NPLSE (897 ± 374 g/ml) (p = 0.2767). Correlation of C3 levels between CSF and serum was weak in NPSLE (r = 0.2885, p = 0.0380), suggesting that up-regulation of C3 in CSF is independent of that in serum. On the other hand, IL-6 levels in CSF of NPSLE (13.4 ± 1233 pg/ml) were significantly higher than that of nonNPSLE (2.14 ± 2.58 pg/ml) (p = 0.0010), which is consistent with the previous observation report about lupus psychosis. It was notable that levels of C3 and IL-6 in CSF showed strong correlation (r = 0.5102, p = 0.0003). This result suggests that C3 and IL-6 in CSF are up-regulated as a result of series of inflammation. Conclusion: We demonstrated the increase of C3 and IL-6 in CSF of NPSLE patients. One possible explanation for the elevation of C3 may be hyper-production of C3 in the brain. Namely, hyper-