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Structural and functional characterization of a novel complement regulatory molecule in rainbow trout (Oncorhynchus mykiss)
Abstracts / Molecular Immunology 44 (2007) 147–266
22 Structural and functional characterization of a novel complement regulatory molecule in rainbow trout (Oncorhynchus mykiss) Hani Boshra a , Tiehui Wang b , Jun Secombes b , J. Oriol Sunyer a
Li a , Christopher
J.
a
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Rosenthal Bldg., Room #413, Philadelphia, PA, USA; b Scottish Fish Immunology Research Center, University of Aberdeen, Aberdeen, Scotland, UK Formation of alternative (C3bBb) and classical (C4b2a) C3 convertases is regulated in part by decay accelerating factor (DAF), C4 binding protein (C4bp) and factor H. These regulatory molecules have in common the ability to accelerate the decay rate of both classical and alternative C3 convertases (DAF, H) thereby inhibiting the cleavage of native C3 into its active form. Unlike mammals, teleost fish are known to have multiple isoforms of C3 that are the products of different genes. Rainbow trout, our fish model, contain three different C3 isoforms, C3-1, C3-3 and C34. Here we have attempted to determine whether regulation of the activation of these C3 molecules is isoform-specific. In this study we have characterized a novel 75 kDa serum protein from rainbow trout (Oncorhynchus mykiss) possessing a unique DAF-like complement regulatory activity. When combined with purified trout factor D and Bf/C2, this molecule inhibited the fluid phase cleavage of trout C3-1, C3-3 and C3-4 in a dose-dependent manner; trout factor B/C2 cleavage however remained unaffected. Cloning of this regulatory protein showed that its sequence displayed the highest degree of homology to factor H molecules from mammalian species. This study suggests that regulation of C3 cleavage in rainbow trout is not isoform specific, although the presence of additional C3-regulatory molecules cannot be ruled out at this point in this species. Finally, this study represents the first structural and functional characterization of a complement regulatory molecule in rainbow trout. doi:10.1016/j.molimm.2006.07.027 23 Mannose-binding lectin (MBL) binding during opsonization is not indicative for its role in phagocytosis N. Brouwer a , D. Zweers a , K.M. Dolman a,b , R. van Zwieten a , M.H.L. Hart a , D. Roos a , T.W. Kuijpers a,b
a Sanquin-AMC Landsteiner Laboratory, Amsterdam, The Netherlands; b Emma Children’s Hospital, AMC, University of Amsterdam, Amsterdam, The Netherlands
The innate host defense molecule MBL functions as an indirect opsonin. Recognition of opsonized micro-organisms by phagocytes occurs via the complement system, which can be activated via the lectin pathway by MBL or ficolin complexed to MBLassociated serine proteases (MASPs). Three mutations in exon 1 of the MBL2 gene lead to deficient MBL serum levels, which results in increased susceptibility to infections. We investigated
157
the role of MBL opsonization in phagocytosis of different microorganisms. Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Candida albicans, Cryptococcus neoformans and zymosan were opsonized with 3% MBL-sufficient or MBLdeficient serum. Opsonized micro-organisms were added to isolated polymorphonuclear leukocytes (PMN) for phagocytosis, or incubated with monoclonal antibodies to assess depositions of various complement components. Both were measured by flow cytometer. The MBL-sufficient sera had a mean MBL concentration of 1.37 g/ml (range 0.54–2.92 g/ml), while the MBL-deficient sera had a mean MBL concentration of 0.06 g/ml (range 0.01–0.25 g/ml). For all micro-organisms tested, the MBL and C3b deposits were significantly higher after opsonization with MBL-sufficient sera than with MBL-deficient sera. Phagocytosis of zymosan (P = 0.003) and Candida albicans (P < 0.05) was lower after opsonization with the MBL-deficient compared to MBL-sufficient sera, whereas no significant difference was detected for the phagocytosis of the various bacteria tested. Thus, the extent of deposited C3b did not correlate with the level of phagocytosis. It seems that MBL is more important in opsonophagocytosis of yeast compared to bacteria and fungi. There is no strict correlation between MBL or C3b deposition and phagocytosis by PMN of the various micro-organisms tested. To further elucidate this phenomenon we will repeat opsonophagocytosis and deposition of several complement components in the presence of blocking ␣C1q F(ab)2 and further studies will be performed to measure the deposition of MASPs on the micro-organisms. Keywords: Mannose-binding lectin; Opsonization; Complement doi:10.1016/j.molimm.2006.07.028 24 Influence of donor C3 allotype on late renal transplantation outcome Katherine M. Brown a , E. Kondeatis a , R.W. Vaughan a , S.P. Kon b , C.K.T. Farmer c , J.D. Taylor a , X. He d , A. Johnston d , C. Horsfield e , B.J.C. Janssen f , P. Gros f , W. Zhou a , S.H. Sacks a , N.S. Sheerin a a
King’s College London, Department of Nephrology and Transplantation, Guy’s Hospital, London, UK; b King’s College Hospital, Denmark Hill, London, UK; c Kent & Canterbury Hospital, Ethelbert Road, Canterbury, UK; d Clinical Pharmacology, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London, UK; e Department of Histopathology, Guy’s and St. Thomas’ NHS Foundation Trust, London, UK; f Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Padualaan, 8 CH Utrecht, The Netherlands There are approximately 2000 renal transplants in the UK per year. Over the last 50 years major advances in immunosuppression have reduced the rate of graft loss due to acute rejection.
22 Structural and functional characterization of a novel complement regulatory molecule in rainbow trout (Oncorhynchus mykiss) Hani Boshra a , Tiehui Wang b , Jun Secombes b , J. Oriol Sunyer a
Li a , Christopher
J.
a
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Rosenthal Bldg., Room #413, Philadelphia, PA, USA; b Scottish Fish Immunology Research Center, University of Aberdeen, Aberdeen, Scotland, UK Formation of alternative (C3bBb) and classical (C4b2a) C3 convertases is regulated in part by decay accelerating factor (DAF), C4 binding protein (C4bp) and factor H. These regulatory molecules have in common the ability to accelerate the decay rate of both classical and alternative C3 convertases (DAF, H) thereby inhibiting the cleavage of native C3 into its active form. Unlike mammals, teleost fish are known to have multiple isoforms of C3 that are the products of different genes. Rainbow trout, our fish model, contain three different C3 isoforms, C3-1, C3-3 and C34. Here we have attempted to determine whether regulation of the activation of these C3 molecules is isoform-specific. In this study we have characterized a novel 75 kDa serum protein from rainbow trout (Oncorhynchus mykiss) possessing a unique DAF-like complement regulatory activity. When combined with purified trout factor D and Bf/C2, this molecule inhibited the fluid phase cleavage of trout C3-1, C3-3 and C3-4 in a dose-dependent manner; trout factor B/C2 cleavage however remained unaffected. Cloning of this regulatory protein showed that its sequence displayed the highest degree of homology to factor H molecules from mammalian species. This study suggests that regulation of C3 cleavage in rainbow trout is not isoform specific, although the presence of additional C3-regulatory molecules cannot be ruled out at this point in this species. Finally, this study represents the first structural and functional characterization of a complement regulatory molecule in rainbow trout. doi:10.1016/j.molimm.2006.07.027 23 Mannose-binding lectin (MBL) binding during opsonization is not indicative for its role in phagocytosis N. Brouwer a , D. Zweers a , K.M. Dolman a,b , R. van Zwieten a , M.H.L. Hart a , D. Roos a , T.W. Kuijpers a,b
a Sanquin-AMC Landsteiner Laboratory, Amsterdam, The Netherlands; b Emma Children’s Hospital, AMC, University of Amsterdam, Amsterdam, The Netherlands
The innate host defense molecule MBL functions as an indirect opsonin. Recognition of opsonized micro-organisms by phagocytes occurs via the complement system, which can be activated via the lectin pathway by MBL or ficolin complexed to MBLassociated serine proteases (MASPs). Three mutations in exon 1 of the MBL2 gene lead to deficient MBL serum levels, which results in increased susceptibility to infections. We investigated
157
the role of MBL opsonization in phagocytosis of different microorganisms. Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Candida albicans, Cryptococcus neoformans and zymosan were opsonized with 3% MBL-sufficient or MBLdeficient serum. Opsonized micro-organisms were added to isolated polymorphonuclear leukocytes (PMN) for phagocytosis, or incubated with monoclonal antibodies to assess depositions of various complement components. Both were measured by flow cytometer. The MBL-sufficient sera had a mean MBL concentration of 1.37 g/ml (range 0.54–2.92 g/ml), while the MBL-deficient sera had a mean MBL concentration of 0.06 g/ml (range 0.01–0.25 g/ml). For all micro-organisms tested, the MBL and C3b deposits were significantly higher after opsonization with MBL-sufficient sera than with MBL-deficient sera. Phagocytosis of zymosan (P = 0.003) and Candida albicans (P < 0.05) was lower after opsonization with the MBL-deficient compared to MBL-sufficient sera, whereas no significant difference was detected for the phagocytosis of the various bacteria tested. Thus, the extent of deposited C3b did not correlate with the level of phagocytosis. It seems that MBL is more important in opsonophagocytosis of yeast compared to bacteria and fungi. There is no strict correlation between MBL or C3b deposition and phagocytosis by PMN of the various micro-organisms tested. To further elucidate this phenomenon we will repeat opsonophagocytosis and deposition of several complement components in the presence of blocking ␣C1q F(ab)2 and further studies will be performed to measure the deposition of MASPs on the micro-organisms. Keywords: Mannose-binding lectin; Opsonization; Complement doi:10.1016/j.molimm.2006.07.028 24 Influence of donor C3 allotype on late renal transplantation outcome Katherine M. Brown a , E. Kondeatis a , R.W. Vaughan a , S.P. Kon b , C.K.T. Farmer c , J.D. Taylor a , X. He d , A. Johnston d , C. Horsfield e , B.J.C. Janssen f , P. Gros f , W. Zhou a , S.H. Sacks a , N.S. Sheerin a a
King’s College London, Department of Nephrology and Transplantation, Guy’s Hospital, London, UK; b King’s College Hospital, Denmark Hill, London, UK; c Kent & Canterbury Hospital, Ethelbert Road, Canterbury, UK; d Clinical Pharmacology, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London, UK; e Department of Histopathology, Guy’s and St. Thomas’ NHS Foundation Trust, London, UK; f Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Padualaan, 8 CH Utrecht, The Netherlands There are approximately 2000 renal transplants in the UK per year. Over the last 50 years major advances in immunosuppression have reduced the rate of graft loss due to acute rejection.