Federici C, Camoin L, Hattab M, Strosberg A D, Couraud P O
Laboratoire d'Immuno-Pharmacologie Moléculaire, ICGM, CNRS UPR 0415, Paris, France.
Eur J Biochem. 1996 May 15;238(1):173-80. doi: 10.1111/j.1432-1033.1996.0173q.x.
To elucidate the molecular mechanisms of the transendothelial migration of leukocytes, we attempted to identify the cellular proteins capable of interaction with the cytoplasmic domain of the intercellular adhesion molecule-1 (ICAM-1) in a rat brain microvessel endothelial cell line (RBE4 cells). A 27-amino-acid synthetic peptide, corresponding to the cytoplasmic domain of rat ICAM-1, was covalently linked to a Sepharose matrix. Upon affinity chromatography of RBE4 cell cytosol, several ICAM-1-interacting proteins were specifically eluted by the soluble peptide. Two of these proteins have been identified by microsequencing as the cytoskeletal protein beta-tubulin and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraP-DH). Experiments carried out with purified GraP-DH or CNBr fragments of GraP-DH indicated that binding to the ICAM-1 matrix was mediated by the C-terminal domain of GraP-DH, containing the binding site of the cofactor NAD+, and that NAD+ could compete with this binding. Using a series of ICAM-1 C-terminal truncated peptides, we could demonstrate that (a) the nitric-oxide-induced covalent linkage of NAD+ to GraP-DH was impaired by these peptides, (b) the glycolytic activity of GraP-DH was drastically inhibited by a truncated peptide containing the 15 C-terminal residues, (c) nitric oxide appeared to prevent this inhibition. Together, our results demonstrate that GraP-DH specifically associates with the isolated ICAM-1 cytoplasmic domain. Since GraP-DH is known as a microtubule bundling protein, these findings suggest that, in a cellular environment, GraP-DH may behave as an adaptor molecule by linking ICAM-1 to the microtubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation.
为了阐明白细胞跨内皮迁移的分子机制，我们试图在大鼠脑微血管内皮细胞系（RBE4细胞）中鉴定能够与细胞间黏附分子1（ICAM-1）胞质结构域相互作用的细胞蛋白。将一段对应于大鼠ICAM-1胞质结构域的27个氨基酸的合成肽共价连接到琼脂糖基质上。对RBE4细胞胞质溶胶进行亲和层析时，几种与ICAM-1相互作用的蛋白被可溶性肽特异性洗脱。其中两种蛋白经微量测序鉴定为细胞骨架蛋白β-微管蛋白和糖酵解酶甘油醛-3-磷酸脱氢酶（GAP-DH）。用纯化的GAP-DH或GAP-DH的溴化氰片段进行的实验表明，与ICAM-1基质的结合是由GAP-DH的C末端结构域介导的，该结构域包含辅因子NAD+的结合位点，并且NAD+可以与这种结合竞争。使用一系列ICAM-1 C末端截短肽，我们可以证明：（a）这些肽会损害一氧化氮诱导的NAD+与GAP-DH的共价连接；（b）含有15个C末端残基的截短肽会大幅抑制GAP-DH的糖酵解活性；（c）一氧化氮似乎可以阻止这种抑制作用。总之，我们的结果表明GAP-DH与分离的ICAM-1胞质结构域特异性结合。由于GAP-DH是一种微管束蛋白，这些发现表明，在细胞环境中，GAP-DH可能通过将ICAM-1与微管网络连接而作为一种衔接分子发挥作用。一氧化氮在调节这种相互作用中的作用值得进一步研究。
