title: A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody.
abstract: Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164-44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6-receptor interaction.
authors.affiliation: Cardiovascular and Metabolic Disease Research, Centocor Inc., 200 Great Valley Parkway, Malvern, PA 19355, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Fisher
authors.forename: Michael
authors.initials: M
authors.lastname: Brigham-Burke
authors.forename: Sheng-Jiun
authors.initials: SJ
authors.lastname: Wu
authors.forename: Jinquan
authors.initials: J
authors.lastname: Luo
authors.forename: Jill
authors.initials: J
authors.lastname: Carton
authors.forename: Kim
authors.initials: K
authors.lastname: Staquet
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.forename: Sheila
authors.initials: S
authors.lastname: Jackson
authors.forename: Deidra
authors.initials: D
authors.lastname: Bethea
authors.forename: Cailin
authors.initials: C
authors.lastname: Chen
authors.forename: Bing
authors.initials: B
authors.lastname: Hu
authors.forename: Jill
authors.initials: J
authors.lastname: Giles-Komar
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D018604:Epitope Mapping; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000099255:Growth Arrest-Specific Protein 6
chemical_list: D000911:Antibodies, Monoclonal; D002118:Calcium; D036341:Intercellular Signaling Peptides and Proteins; D000099255:Growth Arrest-Specific Protein 6