Epitope mapping of function-blocking monoclonal antibody CM6 suggests a "weak" integrin binding site on the laminin-332 LG2 domain.
Epitope mapping of function-blocking monoclonal antibody CM6 suggests a "weak" integrin binding site on the laminin-332 LG2 domain.
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DOI：
10.1002/jcp.22107
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发表时间：
2010-06
影响因子：
5.6
通讯作者：
Quaranta, Vito
中科院分区：
文献类型：
--
作者：
Yamashita, Hironobu;Shang, Meiling;Tripathi, Manisha;Jourquin, Jerome;Georgescu, Walter;Liu, Shanshan;Weidow, Brandy;Quaranta, Vito
层粘连蛋白-332（Ln-332）是一种细胞外基质分子，通过与细胞表面受体（如α3β1和α6β4）相互作用调节细胞粘附、铺展和迁移。在此之前，我们开发了一种针对大鼠Ln-332的功能阻断单克隆抗体CM 6，其阻断Ln-332-α6β4相互作用诱导的半桥粒组装。然而，其在Ln-+332上的表位的位置仍然不清楚。在这项研究中，我们发现CM 6表位位于Ln-332 α3链的LG 2模块上。为了确定该表位中涉及的残基，我们制备了一系列GST融合α3 LG 2突变蛋白，其中通过定点突变策略将大鼠特异性酸替换为人酸。CM 6对这些蛋白质的反应性表明，CM 6与大鼠Ln-332 LG 2模块的1089 NERSVR 1094序列结合。在结构模型中，该序列映射到根据预测暴露于溶剂的LG 2环序列，与其对抗体的可及性一致。CM 6抑制Ln-332上的整联蛋白依赖性细胞粘附，并抑制Ln-332和重组LG 2（rLG 2;但不是rLG 3）上的细胞铺展，表明LG 2上存在α3β1结合位点。然而，我们无法证明rLG 2在标准测定中支持粘附，这表明LG 2可能含有“弱”整联蛋白结合位点，仅在不需要洗涤的扩散测定中可检测到。这些结果与我们之前的发现一起表明，α3β1和α6β4的结合位点在Ln-332 LG结构域中间隔紧密，它们调节替代细胞功能，即粘附/迁移或半桥粒锚定。
Laminin-332 (Ln-332) is an extracellular matrix molecule that regulates cell adhesion, spreading, and migration by interaction with cell surface receptors such as α3β1 and α6β4. Previously, we developed a function-blocking monoclonal antibody against rat Ln-332, CM6, which blocks hemidesmosome assembly induced by Ln-332-α6β4 interactions. However, the location of its epitope on Ln-+332 has remained unclear. In this study, we show that the CM6 epitope is located on the LG2 module of the Ln-332 α3 chain. To specify the residues involved in this epitope, we produced a series of GST-fused α3 LG2 mutant proteins in which rat-specific acids were replaced with human acids by a site-directed mutagenesis strategy. CM6 reactivity against these proteins showed that CM6 binds to the 1089NERSVR1094 sequence of rat Ln-332 LG2 module. In a structural model, this sequence maps to an LG2 loop sequence that is exposed to solvent according to predictions, consistent with its accessibility to antibody. CM6 inhibits integrin-dependent cell adhesion on Ln-332 and inhibits cell spreading on both Ln-332 and recombinant LG2 (rLG2; but not rLG3), suggesting the presence of an α3β1 binding site on LG2. However, we were unable to show that rLG2 supports adhesion in standard assays, suggesting that LG2 may contain a “weak” integrin binding site, only detectable in spreading assays that do not require washes. These results, together with our previous findings, indicate that binding sites for α3β1 and α6β4 are closely spaced in the Ln-332 LG domains where they regulate alternative cell functions, namely adhesion/migration or hemidesmosome anchoring.
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影响因子：
4.8
作者：
Nguyen, BP;Ren, XD;Carter, WG
通讯作者：
Carter, WG
影响因子：
4.8
作者：
Ido, Hiroyuki;Nakamura, Aya;Sekiguchi, Kiyotoshi
通讯作者：
Sekiguchi, Kiyotoshi
DOI：
10.1006/bbrc.1998.9400
发表时间：
1998-10-09
影响因子：
3.1
作者：
Falk-Marzillier, J;Domanico, SZ;Quaranta, V
通讯作者：
Quaranta, V
影响因子：
4
作者：
Manohar, A;Shome, SG;DiPersio, CM
通讯作者：
DiPersio, CM
DOI：
10.1074/jbc.m109.026658
发表时间：
2009-08-21
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Carafoli F;Clout NJ;Hohenester E
通讯作者：
Hohenester E