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Interaction of mannan-binding lectin with IgA
83
23% Control
of the MBL pathway of complement activation
Steen Vang
Petersen, St&en Thiel, Thomas Vorup-Jensen, Lisbeth Jensen, and Jens Christian Jensenius. Department of Medical Microbiology of Aarhus, Denmark
and Immunology, University
The first component of the MBL pathway of complement activation (the MBL-complex) consists of the carbohydrate binding protein mammn-binding lectin (MBL), two associated serine proteases MASP-1 and MASP-2 (mannan-binding lectin associated serine protease- and-2). and a fourth protein, MApl9, which is an alternative spliced product of the MASP-2 gene. Upon binding to carbohydrate structures on the surfaces of microorganisms, the MBL-complex initiates complement activation by cleavage of complement component C4. Activated C4 binds covalently as C4b to nearby amino and hydroxyl groups. A solid phase mannan-coated surface thus provides for a simple assay of the biological activity. Through manipulation of the conditmns this assay can be made selective for the MBL pathway. Using such an assay we have evaluated the inhibitory capacity of various biological and synthetic protease inhibitors towards the MBL-complex. In parallel, the inhibitory effect on the classical pathway of complement activation was estimated using an assay based on IgG-coated surfaces. We find that the two pathways differ markedly in their susceptibility to inhibition by synthetic compounds. Considering biological inhibitors, both pathways are inhibited by Cl inhibitor and alpha-2.macroglobulin. Trasylol (aprotinin), was found to inhibit selectively the MBL pathway. Such analyses may prove useful when considering therapeutic applications of inhibitors in the control of inflammatory reactions trickered by either pathway. lectin with IgA 233- Interaction of manna-binding Anja Roes, DanBlle J. van Gijlswijk-Janssen, Ria Faber-Krol and Mohamed R. Daba, Department ofNephrology, Leiden UniversiQ Medical Center, L&en, the Netherlands. the l&in pathway of complement activation, initiated by binding of mannan-binding lectin (MBL) to one of its ligands, is proposed to have a key role in innate immunity. Recent studies suggest the presence of MBL in glomerular complement deposits, a.o. in IgA nephropathy. IgA nephropathy is the most common primary glomerular disease worldwide. Mesangial deposition of IgA together with complement deposition from C3 until C9 leads to end stage renal failure in most cases. In view of the controversy considering the capacity of IgA to activate complement and the mechanisms involved, we have now analyzed the interaction of IgA with MBL. Purified human MBL bound to human IgA in a dose- and calcium-dependent manner, as shown by ELISA. Incubation of MBL in the presence of mannose abolished its binding to IgA, indicating involvement of the &tin-domain of MBL in binding to IgA. Similar features were observed concerning the binding of MBL to mannan. Co-incubation of MBL with IgA dose-dependently inhibited binding of MBL to mannan, indicating an interaction between MBL and IgA in the fluid phase. MBL was purified by affinity chromatography using immobilized mannan. ELISA and Western blotting demonstrated the presence of IgA in these MBL-preparations. Furthermore, ELISA experiments strongly suggested the presence of MBL-IgA complexes in 5 out of 5 MBL preparations obtained from healthy donors. These studies demonstrate for the first time that MBL is able to interact with IgA. Since MBL is able to initiate activation of C4, our studies provide a possible explanation for the observed mesangial deposition of IgA in association with C4 and CCbinding protein hut not with Clq, in patients with IgA nephropatby. Thus, the MBL route of complement activation may be responsible for the induction of the inflammatory response associated with IgA nephropathy.
234- C4b-BINDING PROTEIN AND MANNOSE BINDING LECTlN INTERACTIONS WITH l32-GLYCOPROTEIN I: ASSOCIATION OF COMPLEMENT ACTIVATION AND ANTIPHOSPHOLIPID ANTIBODIES. Derek S. Sim’, Steffen Thiel’, and Dana V. Devine’.2. ‘Dept of Biochemistry and 2Pathology, U. of British Columbia, Vancouver, Canada, ‘Dept of Medical Microbiology and Immunology, U. of Aarhus, Denmark. 02-glycoprotein I (l32GPI) is a major target of antiphospholipid antibodies (APL) in antiphospholipid syndrome (APS). Complement consumption has been reported to be associated with APL. C4b-binding protein (C4BP) interacted with BZGPI-coated ELISA plate through the R-chain on C4BP. C4b did not inhibit this C4BP-B2GPl binding. APL IgG inhibited this RZGPl-C4BP interaction. Mannose binding lectin (MBL) also interacted directly with R2GPI-coated ELISA plate at 2.5 mM CaC&. This interaction was inhibited by the addition of EDTA or N-acetylmannosamine. APL IgG significantly increased MBL binding to DZGPI suggesting that MBL had affinity towards aPL-l32GPI complex. 82GPI-coated ELISA wells were used to capture aPL from patient sera to form aPL-l32GPI complexes to assess APL-mediated C5b-9 generation. Samples with complement activation levels higher than the normal mean + 3 SD were considered as positive. Complement activation was detected in 47.5 % (19/40) of patients. The level of complement activation and the level of anti- l32GPI IgM showed significant correlation (p
235 CHARACTERIZATION OF MONOCLONAL ANTIBODIES (mAb) AGAINST NATIVE AND RECOMBINANT HUMAN MANNOSE-BINDING LECTIN (MBL) H. Zhao and G.L.Stahl. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. We have recently demonstrated that MBL plays an important role in complement activation following endothelial oxidative stress. We have generated a panel of functional (3F8, hMBL1.2 and 2A9) and non-functional (IClO) mAb against human MBL and have begun characterization of the binding domains. All antibodies recognize recombinant (r) and native (n) non-denatured/reduced MBL by western analysis. 2A9 and hMBL1.2 easily recognize reduced/ denatured recombinant or native MBL. Recombinant MBL bound to a Biacore Cl chip recognized and bound mannan. Biacore analysis demonstrated that the l$ for 3F6, hMBLl.2 and 2A9 was 2.63x10-’ M; 1.39x10’ M; 5.66xlU’ M respectively. The k. for 3F8, hMBLl.2 and 2A9 was 2.7 x105 M” S.‘; 3.6 xl O4 M” s”; 1.22~10s M“ s.’ respectively. The kd for 3F8, hMBL1.2 and 2A9 was 7.O9x1O.4 s”; 5.05x10-~ s-‘; 6.87 ~10.~ s-’ respectively. In addition, Biacore epitope mapping showed that the antibodies recognized different epitopes. Digestion of rMBL with ArgC followed by western analysis under reduced conditions with 2A9 demonstrated a single band of - 21kD MW, corresponding to amino acids 122-248 within MBL. 1 Cl0 recognized a similar band under non-reduced conditions but failed to recognize this band under reduced conditions. These data suggest that the disulfide linkage between cysteinel55 and 244 might be necessary for lCl0 to recognize MBL. Further characterization of the binding epitopes of these mAb may yield important information on the carbohydrate recognition domain of MBL.
23% Control
of the MBL pathway of complement activation
Steen Vang
Petersen, St&en Thiel, Thomas Vorup-Jensen, Lisbeth Jensen, and Jens Christian Jensenius. Department of Medical Microbiology of Aarhus, Denmark
and Immunology, University
The first component of the MBL pathway of complement activation (the MBL-complex) consists of the carbohydrate binding protein mammn-binding lectin (MBL), two associated serine proteases MASP-1 and MASP-2 (mannan-binding lectin associated serine protease- and-2). and a fourth protein, MApl9, which is an alternative spliced product of the MASP-2 gene. Upon binding to carbohydrate structures on the surfaces of microorganisms, the MBL-complex initiates complement activation by cleavage of complement component C4. Activated C4 binds covalently as C4b to nearby amino and hydroxyl groups. A solid phase mannan-coated surface thus provides for a simple assay of the biological activity. Through manipulation of the conditmns this assay can be made selective for the MBL pathway. Using such an assay we have evaluated the inhibitory capacity of various biological and synthetic protease inhibitors towards the MBL-complex. In parallel, the inhibitory effect on the classical pathway of complement activation was estimated using an assay based on IgG-coated surfaces. We find that the two pathways differ markedly in their susceptibility to inhibition by synthetic compounds. Considering biological inhibitors, both pathways are inhibited by Cl inhibitor and alpha-2.macroglobulin. Trasylol (aprotinin), was found to inhibit selectively the MBL pathway. Such analyses may prove useful when considering therapeutic applications of inhibitors in the control of inflammatory reactions trickered by either pathway. lectin with IgA 233- Interaction of manna-binding Anja Roes, DanBlle J. van Gijlswijk-Janssen, Ria Faber-Krol and Mohamed R. Daba, Department ofNephrology, Leiden UniversiQ Medical Center, L&en, the Netherlands. the l&in pathway of complement activation, initiated by binding of mannan-binding lectin (MBL) to one of its ligands, is proposed to have a key role in innate immunity. Recent studies suggest the presence of MBL in glomerular complement deposits, a.o. in IgA nephropathy. IgA nephropathy is the most common primary glomerular disease worldwide. Mesangial deposition of IgA together with complement deposition from C3 until C9 leads to end stage renal failure in most cases. In view of the controversy considering the capacity of IgA to activate complement and the mechanisms involved, we have now analyzed the interaction of IgA with MBL. Purified human MBL bound to human IgA in a dose- and calcium-dependent manner, as shown by ELISA. Incubation of MBL in the presence of mannose abolished its binding to IgA, indicating involvement of the &tin-domain of MBL in binding to IgA. Similar features were observed concerning the binding of MBL to mannan. Co-incubation of MBL with IgA dose-dependently inhibited binding of MBL to mannan, indicating an interaction between MBL and IgA in the fluid phase. MBL was purified by affinity chromatography using immobilized mannan. ELISA and Western blotting demonstrated the presence of IgA in these MBL-preparations. Furthermore, ELISA experiments strongly suggested the presence of MBL-IgA complexes in 5 out of 5 MBL preparations obtained from healthy donors. These studies demonstrate for the first time that MBL is able to interact with IgA. Since MBL is able to initiate activation of C4, our studies provide a possible explanation for the observed mesangial deposition of IgA in association with C4 and CCbinding protein hut not with Clq, in patients with IgA nephropatby. Thus, the MBL route of complement activation may be responsible for the induction of the inflammatory response associated with IgA nephropathy.
234- C4b-BINDING PROTEIN AND MANNOSE BINDING LECTlN INTERACTIONS WITH l32-GLYCOPROTEIN I: ASSOCIATION OF COMPLEMENT ACTIVATION AND ANTIPHOSPHOLIPID ANTIBODIES. Derek S. Sim’, Steffen Thiel’, and Dana V. Devine’.2. ‘Dept of Biochemistry and 2Pathology, U. of British Columbia, Vancouver, Canada, ‘Dept of Medical Microbiology and Immunology, U. of Aarhus, Denmark. 02-glycoprotein I (l32GPI) is a major target of antiphospholipid antibodies (APL) in antiphospholipid syndrome (APS). Complement consumption has been reported to be associated with APL. C4b-binding protein (C4BP) interacted with BZGPI-coated ELISA plate through the R-chain on C4BP. C4b did not inhibit this C4BP-B2GPl binding. APL IgG inhibited this RZGPl-C4BP interaction. Mannose binding lectin (MBL) also interacted directly with R2GPI-coated ELISA plate at 2.5 mM CaC&. This interaction was inhibited by the addition of EDTA or N-acetylmannosamine. APL IgG significantly increased MBL binding to DZGPI suggesting that MBL had affinity towards aPL-l32GPI complex. 82GPI-coated ELISA wells were used to capture aPL from patient sera to form aPL-l32GPI complexes to assess APL-mediated C5b-9 generation. Samples with complement activation levels higher than the normal mean + 3 SD were considered as positive. Complement activation was detected in 47.5 % (19/40) of patients. The level of complement activation and the level of anti- l32GPI IgM showed significant correlation (p
235 CHARACTERIZATION OF MONOCLONAL ANTIBODIES (mAb) AGAINST NATIVE AND RECOMBINANT HUMAN MANNOSE-BINDING LECTIN (MBL) H. Zhao and G.L.Stahl. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. We have recently demonstrated that MBL plays an important role in complement activation following endothelial oxidative stress. We have generated a panel of functional (3F8, hMBL1.2 and 2A9) and non-functional (IClO) mAb against human MBL and have begun characterization of the binding domains. All antibodies recognize recombinant (r) and native (n) non-denatured/reduced MBL by western analysis. 2A9 and hMBL1.2 easily recognize reduced/ denatured recombinant or native MBL. Recombinant MBL bound to a Biacore Cl chip recognized and bound mannan. Biacore analysis demonstrated that the l$ for 3F6, hMBLl.2 and 2A9 was 2.63x10-’ M; 1.39x10’ M; 5.66xlU’ M respectively. The k. for 3F8, hMBLl.2 and 2A9 was 2.7 x105 M” S.‘; 3.6 xl O4 M” s”; 1.22~10s M“ s.’ respectively. The kd for 3F8, hMBL1.2 and 2A9 was 7.O9x1O.4 s”; 5.05x10-~ s-‘; 6.87 ~10.~ s-’ respectively. In addition, Biacore epitope mapping showed that the antibodies recognized different epitopes. Digestion of rMBL with ArgC followed by western analysis under reduced conditions with 2A9 demonstrated a single band of - 21kD MW, corresponding to amino acids 122-248 within MBL. 1 Cl0 recognized a similar band under non-reduced conditions but failed to recognize this band under reduced conditions. These data suggest that the disulfide linkage between cysteinel55 and 244 might be necessary for lCl0 to recognize MBL. Further characterization of the binding epitopes of these mAb may yield important information on the carbohydrate recognition domain of MBL.