Binding sites of leukocyte β2 integrins (LFA-1, Mac-1) on the human ICAM-4/LW blood group protein
Binding sites of leukocyte β2 integrins (LFA-1, Mac-1) on the human ICAM-4/LW blood group protein
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DOI：
10.1074/jbc.m002823200
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发表时间：
2000-08-25
影响因子：
4.8
通讯作者：
Bailly, P
中科院分区：
文献类型：
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作者：
Hermand, P;Huet, M;Bailly, P
红细胞ICAM-4/LW血型糖蛋白属于细胞间粘附分子（ICAM）家族，据报道与CD 11 a/CD 18（LFA-1）和CD 11b/CD 18（Mac-1）β（2）整合素相互作用。为了更好地确定ICAM-4/β 2整联蛋白相互作用的基础，我们已经产生了野生型、结构域缺失和突变的重组嵌合ICAM-4-Fc蛋白，并在细胞粘附测定中分析了它们与LFA-1和Mac-1 L细胞稳定转染子的相互作用。我们发现，抗CD 11 a、CD 11b、CD 18或LWab的单克隆抗体阻断转染L细胞与固定化ICAM-4-Fc蛋白的粘附，并且ICAM-4/β 2整联蛋白相互作用对二价阳离子Ca 2+和Mg 2+的存在高度敏感。ICAM-8细胞外部分的单个Ig结构域D1或D2的缺失表明LFA-1结合第一Ig样结构域，而Mac-1结合位点包括第一和第二Ig样结构域。基于ICAM-8的晶体结构，我们提出了ICAM-4的Ig样结构域D1和D2的模型。因此，通过在ICAM-4分子的所有表面上散布的22个氨基酸位置的定点诱变，我们鉴定了四个暴露的残基，在CFG表面上的Leu（80）、Trp（93）和Arg（97）和在结构域D1的E-F环上的Trp（77），其可能作为结合位点的一部分接触LFA-1。然而，结构域D1的CFG面上的单突变体R52 E和双突变体T91 Q（对应于ICAM-1与LFA-1结合的关键残基Glu（34）和Gln（73））对LFA-1结合没有影响。相反，结构域D1的CFG面上的所有突变体和结构域D2的C ′-E环中的残基Glu（151）和Thr（154）似乎在Mac-1结合中起主导作用。这些数据表明ICAM-4上LFA-1的结合位点与Mac-1的结合位点重叠但不同。
The red cell ICAM-4/LW blood group glycoprotein, which belongs to the family of intercellular adhesion molecules (ICAMs), has been reported to interact with CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) beta(2) integrins. To better define the basis of the ICAM-4/beta(2) integrin interaction, we have generated wild-type, domain-deleted and mutated recombinant chimeric ICAM-4-Fc proteins and analyzed their interaction in a cellular adhesion assay with LFA-1 and Mac-1 L-cell stable transfectants. We found that monoclonal antibodies against CD11a, CD11b, CD18, or LWab block adhesion of transfectant L-cells to immobilized ICAM-4-Fc protein and that the ICAM-4/beta(2) integrin interaction was highly sensitive to the presence of the divalent cations Ca2+ and Mg2+. Deletion of individual Ig-domains D1 or D2 of the extracellular part of ICAM-8 showed that LFA-1 binds to the first Ig-like domain, whereas the Mac-1 binding site encompassed both the first and the second Ig-like domains. Based on the crystal structure of ICAM-8, we propose a model for the Ig-like domains D1 and D2 of ICAM-4. Accordingly, by site-directed mutagenesis of 22 amino acid positions spread out on all faces of the ICAM-4 molecule, we identified four exposed residues, Leu(80), Trp(93), and Arg(97) On the CFG face and Trp(77) On the E-F loop of domain D1 that may contact LFA-1 as part of the binding site. However, the single and double mutants R52E and T91Q on the CFG face of domain D1, which correspond to the key residues Glu(34) and Gln(73) for ICAM-1 binding to LFA-1, had no effect on LFA-1 binding. In contrast, all mutants on the CFG face of domain D1 and residues Glu(151) and Thr(154) in the C'-E loop of the domain D2 seem to play a dominant role in Mac-1 binding. These data suggest that the binding site for LFA-1 on ICAM-4 overlaps but is distinct from the Mac-1 binding site.