Identification of human mannose binding lectin (MBL) recognition sites for novel inhibitory antibodies.

Mannose binding lectin (MBL) binding initiates activation of the lectin complement pathway. Recent studies from our laboratory have demonstrated that MBL-dependent complement activation mediates cellular injury following oxidative stress in vivo and in vitro. A panel of novel inhibitory monoclonal antibodies (MAbs) against MBL (e.g., MAb 3F8, 2A9, and hMBL1.2) has been developed that inhibit MBL binding and lectin pathway activation. Here, we further characterized the interactions of these MAbs and their Fab fragments to MBL. Whole MAbs or their Fab fragments bound to MBL with relatively high affinity. Fab fragments of 3F8 were functionally effective in inhibiting MBL-dependent complement activation, however, steric hindrance of MAb 2A9 was essential for inhibition of MBL-dependent complement activation. We identified the hinge region, and residues EDCVLLL within the carbohydrate recognition domain of MBL as the recognition sites for MAb 3F8 and 2A9, respectively. The interaction of MAbs (e.g., 3F8 and 2A9) to MBL was dependent on the conformation of their recognition sites. These findings demonstrate that MBL binding can be inhibited by at least two separate and independent mechanisms.

[1]  T. Kase,et al.  Characterization of truncated human mannan-binding protein (MBP) expressed in Escherichia coli. , 1998, Bioscience, biotechnology, and biochemistry.

[2]  K. Drickamer,et al.  Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein. , 1986, The Journal of biological chemistry.

[3]  T. Fujita,et al.  Glomerular deposition of mannose-binding lectin (MBL) indicates a novel mechanism of complement activation in IgA nephropathy. , 1998, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[4]  S. Meri,et al.  Complement activation after oxidative stress: role of the lectin complement pathway. , 2000, The American journal of pathology.

[5]  T. Kawasaki,et al.  A serum lectin (mannan-binding protein) has complement-dependent bactericidal activity. , 1989, Journal of biochemistry.

[6]  D. Bhole,et al.  Therapeutic potential of targeting the complement cascade in critical care medicine. , 2003, Critical Care Medicine.

[7]  M. Weiser,et al.  Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement , 1996, The Journal of experimental medicine.

[8]  R. Timpl,et al.  Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q. , 1990, Journal of immunology.

[9]  A. Agah,et al.  Complement activation following oxidative stress. , 1999, Molecular immunology.

[10]  S. Sheriff,et al.  The collectins in innate immunity. , 1996, Current opinion in immunology.

[11]  G. R. Carson,et al.  Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. , 1990, Science.

[12]  Rebecca L Rich,et al.  A survey of the year 2002 commercial optical biosensor literature , 2003, Journal of molecular recognition : JMR.

[13]  A. Tenner,et al.  Human Cord Blood Leukocyte Innate Immune Responses to Defense Collagens , 2003, Pediatric Research.

[14]  J. Mccoy,et al.  Displaying libraries of conformationally constrained peptides on the surface of Escherichia coli as flagellin fusions. , 1998, Methods in molecular biology.

[15]  A second serine protease associated with mannan-binding lectin that activates complement , 1997 .

[16]  M. Turner,et al.  The level of mannan‐binding protein regulates the binding of complement‐derived opsonins to mannan and zymosan at low serum concentrations , 1990, Clinical and experimental immunology.

[17]  R. Ezekowitz,et al.  A human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins [published erratum appears in J Exp Med 1991 Sep 1;174(3):753] , 1988, The Journal of experimental medicine.

[18]  K. Drickamer,et al.  Two distinct classes of carbohydrate-recognition domains in animal lectins. , 1988, The Journal of biological chemistry.

[19]  Wayne A. Hendrickson,et al.  Structure of a C-type mannose-binding protein complexed with an oligosaccharide , 1992, Nature.

[20]  Yuan-chuan Lee,et al.  Difference in binding-site architecture of the serum-type and liver-type mannose-binding proteins , 1997, Glycoconjugate Journal.

[21]  W. Reenstra,et al.  Ulex europaeus agglutinin II (UEA‐II) is a novel, potent inhibitor of complement activation , 2001, Protein science : a publication of the Protein Society.

[22]  Hansjörg Hoppe,et al.  Collectins — soluble proteins containing collagenous regions and lectin domains — and their roles in innate immunity , 1994, Protein science : a publication of the Protein Society.

[23]  S. Yamamoto,et al.  High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells. , 1999, Journal of immunological methods.

[24]  L. Matis,et al.  Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal complement components and inhibition by anti-C5 therapy. , 1998, Circulation.

[25]  A. Beavil,et al.  Alpha-helical coiled-coil stalks in the low-affinity receptor for IgE (Fc epsilon RII/CD23) and related C-type lectins. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[26]  T. Kawasaki,et al.  Isolation and characterization of a mannan-binding protein from human serum. , 1983, Journal of biochemistry.

[27]  J. Schweinle,et al.  Distinct and overlapping functions of allelic forms of human mannose binding protein , 1992, Nature genetics.

[28]  Mark C. Surles,et al.  Sculpting proteins interactively: Continual energy minimization embedded in a graphical modeling system , 1994, Protein science : a publication of the Protein Society.

[29]  T. Fujita,et al.  Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease , 1992, The Journal of experimental medicine.

[30]  T. Fujita,et al.  Cutting Edge: Complement-Activating Complex of Ficolin and Mannose-Binding Lectin-Associated Serine Protease1 , 2000, The Journal of Immunology.

[31]  R. Würzner,et al.  Glomerular deposition of mannose-binding lectin in human glomerulonephritis. , 1999, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[32]  T. Fujita,et al.  The lectin‐complement pathway – its role in innate immunity and evolution , 2004, Immunological reviews.

[33]  S. Sheriff,et al.  Human mannose-binding protein carbohydrate recognition domain trimerizes through a triple α-helical coiled-coil , 1994, Nature Structural Biology.

[34]  T. Fujita,et al.  Complement activation through the lectin pathway in patients with Henoch-Schönlein purpura nephritis. , 2000, American journal of kidney diseases : the official journal of the National Kidney Foundation.

[35]  Michael C. Montalto,et al.  A Keratin Peptide Inhibits Mannose-Binding Lectin1 , 2001, The Journal of Immunology.

[36]  S. Rendig,et al.  Limitation of reperfusion injury by a monoclonal antibody to C5a during myocardial infarction in pigs. , 1995, The American journal of physiology.

[37]  Michael C. Montalto,et al.  Inhibition of Mannose-Binding Lectin Reduces Postischemic Myocardial Reperfusion Injury , 2001, Circulation.

[38]  W. Weis,et al.  Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing. , 1991, Science.