A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody
A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody
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DOI：
10.1042/bj20040859
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发表时间：
2005-05-01
影响因子：
4.1
通讯作者：
Yang, J
中科院分区：
文献类型：
--
作者：
Fisher, PW;Brigham-Burke, M;Yang, J
Gas 6（生长抑制特异性基因6）是一种维生素K依赖性蛋白，已知可激活Ax 1家族的受体酪氨酸激酶。它是血栓形成和许多其他生物功能的重要调节剂。Gas 6的C-末端与受体结合，由两个层粘连蛋白样球状结构域LG 1和LG 2组成。据报道，LG 1和LG 2结构域连接处的Ca 2+结合位点和LG 2结构域处的疏水片对于受体结合是重要的[Sasaki，Knyazev，Cheburkin，Gohring，Tisi，Ullrich，Timpl和Hohenester（2002）J.Biol.Chem.277，44164-44170]。在本研究中，我们开发了一种针对Gas 6的中和人单克隆抗体，命名为CNTO 300。通过用重组人Gas 6蛋白免疫表达人IgG的转基因小鼠产生抗体，并且从不稳定的杂交瘤克隆中拯救抗Gas 6 IgG序列。CNTO 300抗体以剂量依赖性方式部分抑制Gas 6与其受体的结合。为了进一步表征Gas 6与该抗体之间的相互作用，将CNTO 300与重组Gas 6的结合动力学与独立表达的LG 1和LG 2进行比较。CNTO 300抗体显示出与Gas 6和LG 1相当的结合亲和力，但对Ca 2+的依赖性不同。未检测到与LG 2的结合。在EDTA存在下，抗体与Gas 6的结合被破坏，但EDTA对LG 1结合没有明显的影响。进一步的表位作图鉴定了CNTO 300抗体识别的Gas 6肽序列。发现该肽序列位于远离Ca 2+结合位点和疏水补丁的LG 1结构域。通过BIAcore分析检测到Gas 6与其受体和CNTO 300抗体的共相互作用，表明LG 1结构域上存在第二个受体结合位点。Gas 6受体与独立表达的LG 1结构域的直接结合进一步支持了这一假设。我们的研究结果首次揭示了Gas 6-受体相互作用的第二个结合位点。
Gas6 (growth-affest- specific gene 6) is a vitamin K-dependent protein known to activate the Ax1 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.