van der Merwe P A, Barclay A N, Mason D W, Davies E A, Morgan B P, Tone M, Krishnam A K, Ianelli C, Davis S J
MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, U.K.
Biochemistry. 1994 Aug 23;33(33):10149-60. doi: 10.1021/bi00199a043.
CD2 is a T lymphocyte cell-adhesion molecule (CAM) belonging to the immunoglobulin superfamily (IgSF) which mediates transient adhesion of T cells to antigen-presenting cells and target cells. Reported ligands for human CD2 include the structurally-related IgSF CAMs CD58 (LFA-3) and CD48 as well as, more controversially, the unrelated cell-surface glycoprotein CD59. Using surface plasmon resonance technology, which avoids several pitfalls of conventional binding assays, we recently reported that rat CD2 binds rat CD48 with a very low affinity (Kd 60-90 microM) and dissociates rapidly (koff > or = 6 s-1) [van der Merwe, P. A., Brown, M. H., Davis, S. J., & Barclay, A. N. (1993) EMBO J. 12, 4945-4954]. In contrast, a study using conventional equilibrium binding methods reported a much higher affinity (Kd 0.4 microM) for human CD2 binding CD58 which suggested that the weak binding of rat CD2 to CD48 may not represent a typical CAM interaction. In the present study we have used surface plasmon resonance to obtain definitive affinity and kinetic data on the interactions of a soluble, recombinant form of human CD2 with soluble forms of CD58, CD48, and CD59. Binding of CD2 to CD58 was readily detected but we were unable to detect any direct interaction between CD2 and either CD59 or CD48 under conditions in which very low affinity interactions (Kd approximately 0.5 mM) would have been detected. In contrast to previous reports we found that human CD2 bound CD58 with a very low affinity (Kd 9-22 microM) and dissociated with an extremely fast dissociation rate constant (koff > or = 4 s-1). The association rate constant (kon) could not be measured directly but was calculated to be > or = 400,000 M-1s-1. Taken together, these results provide conclusive evidence that CAM interactions can have very low affinities and extremely fast dissociation rate constants.
CD2是一种属于免疫球蛋白超家族（IgSF）的T淋巴细胞细胞黏附分子（CAM），它介导T细胞与抗原呈递细胞及靶细胞的瞬时黏附。据报道，人CD2的配体包括结构相关的IgSF CAMs CD58（淋巴细胞功能相关抗原-3，LFA-3）和CD48，以及更具争议性的非相关细胞表面糖蛋白CD59。利用表面等离子体共振技术（该技术避免了传统结合测定的几个缺陷），我们最近报道大鼠CD2与大鼠CD48以非常低的亲和力（解离常数Kd为60 - 90微摩尔）结合且快速解离（解离速率常数koff≥6 s-1）[范德默韦，P. A.，布朗，M. H.，戴维斯，S. J.，& 巴克利，A. N.（1993年）《欧洲分子生物学组织杂志》第12卷，4945 - 4954页]。相比之下，一项使用传统平衡结合方法的研究报道人CD2与CD58结合的亲和力要高得多（Kd为0.4微摩尔），这表明大鼠CD2与CD48的弱结合可能并不代表典型的CAM相互作用。在本研究中，我们利用表面等离子体共振技术获得了关于可溶性重组形式的人CD2与可溶性形式的CD58、CD48和CD59相互作用的确切亲和力和动力学数据。很容易检测到CD2与CD58的结合，但在能够检测到非常低亲和力相互作用（Kd约为(0.5)毫摩尔）的条件下，我们未能检测到CD2与CD59或CD48之间的任何直接相互作用。与先前的报道相反，我们发现人CD2与CD58以非常低的亲和力（Kd为9 - 22微摩尔）结合且以极快的解离速率常数解离（koff≥4 s-1）。结合速率常数（kon）无法直接测量，但经计算≥(400,000) M-1s-1。综上所述，这些结果提供了确凿证据，表明CAM相互作用可以具有非常低的亲和力和极快的解离速率常数。
