Blanco J, Marié I, Callebaut C, Jacotot E, Krust B, Hovanessian A G
Unité de virologie et d'Immunologie Cellulaire, UA CNRS 1157, Institut Pasteur, Paris Cedex, France.
Exp Cell Res. 1996 May 25;225(1):102-11. doi: 10.1006/excr.1996.0161.
Adenosine deaminase (ADA) and the HIV-1 transactivator protein Tat have been reported to bind to human CD26, also known as dipeptidyl peptidase IV (DPP IV). In order to demonstrate the specificity of such binding under native conditions of CD26, i.e., when expressed on the cell surface, we established murine cell lines expressing transfected human CD26, either wild-type or mutated at its serine-630, which inactivates the DPP IV activity. This experimental system is advantageous since murine ADA does not bind human CD26, whereas human and bovine ADA bind. Consequently, murine cell clones expressing either the wild-type or mutated form of human CD26 were found to bind specifically bovine 125I-labeled ADA with a high affinity (KD = 12 +/- 2 nM and 11 +/- 4 nM, respectively). No specific binding of 125I-labeled ADA was observed to murine clones not expressing human CD26. The binding of 125I-labeled ADA to CD26 was further characterized by the use of monoclonal antibodies specific to human CD26. The results obtained were in accord with those reported previously using other experimental models. These observations indicated that the murine cells expressing human CD26 provide a highly suitable model to investigate the potential binding of HIV-1 Tat to CD26. In contrast to previously published results, however, we could not demonstrate a specific interaction between Tat and human CD26. The 125I-labeled ADA-specific binding to human CD26 was not affected by Tat, even at concentrations which induced cell death. Similarly, the binding of several monoclonal antibodies to human CD26 was not modified by the addition of Tat. More significantly, Tat binding to different murine cell clones (human CD26 negative or positive) was found not to be correlated with the expression of human CD26. Finally, the toxic effect of Tat on the growth of different murine cell clones was independent of human CD26 expression. Taken together, these observations further confirm the specific binding of ADA to human CD26 and point out that CD26 is not the target of HIV-1 Tat protein.
据报道，腺苷脱氨酶（ADA）和HIV-1反式激活蛋白Tat可与人CD26结合，CD26也被称为二肽基肽酶IV（DPP IV）。为了在CD26的天然条件下，即当它在细胞表面表达时，证明这种结合的特异性，我们建立了表达转染人CD26的小鼠细胞系，该CD26可以是野生型，也可以是丝氨酸-630位点发生突变的类型，丝氨酸-630位点突变会使DPP IV活性失活。这个实验系统具有优势，因为小鼠ADA不与人CD26结合，而人和牛的ADA则会结合。因此，发现表达野生型或突变型人CD26的小鼠细胞克隆能够以高亲和力特异性结合牛的125I标记的ADA（KD分别为12±2 nM和11±4 nM）。未观察到125I标记的ADA与不表达人CD26的小鼠克隆有特异性结合。通过使用对人CD26特异的单克隆抗体，进一步对125I标记的ADA与人CD26的结合进行了表征。得到的结果与先前使用其他实验模型报道的结果一致。这些观察结果表明，表达人CD26的小鼠细胞为研究HIV-1 Tat与CD26的潜在结合提供了一个非常合适的模型。然而，与先前发表的结果相反，我们未能证明Tat与人CD26之间存在特异性相互作用。即使在诱导细胞死亡的浓度下，125I标记的ADA与人CD26的特异性结合也不受Tat的影响。同样，几种单克隆抗体与人CD26的结合也不会因添加Tat而改变。更重要的是，发现Tat与不同小鼠细胞克隆（人CD26阴性或阳性）的结合与人CD26的表达无关。最后，Tat对不同小鼠细胞克隆生长的毒性作用与人CD26的表达无关。综上所述，这些观察结果进一步证实了ADA与人CD26的特异性结合，并指出CD26不是HIV-1 Tat蛋白的靶标。
