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.
Zhao
Hui
H
Center for Experimental Therapeutics & Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
Wakamiya
Nobutaka
N
Suzuki
Yasuhiko
Y
Hamonko
Matthew T
MT
Stahl
Gregory L
GL
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D037222:Collectins; D003167:Complement Activation; D000939:Epitopes / Q000737:chemistry; D017353:Gene Deletion; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018384:Oxidative Stress; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D002352:Carrier Proteins; D037222:Collectins; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins