Dustin M L
Center for Immunology and the Department of Pathology, Washington University School of Medicine, Box 8118, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 Jun 20;272(25):15782-8. doi: 10.1074/jbc.272.25.15782.
The interaction of the T cell glycoprotein CD2 and its ligand CD58 is important for T cell interaction with antigen-presenting and target cells. The binding interaction is of low affinity and has a fast off-rate (>5 s-1) in solution. However, solution measurements may not accurately predict the behavior of molecules in an adhesive contact area. Interaction between T cells that express CD2 and glass-supported planar bilayers containing purified and fluorescently labeled CD58 leads to accumulation of CD58 (fluorescence) in the cell/bilayer contact area. CD58 molecules accumulated within the contact area in excess of the CD58 density in the bilayer outside the contact area can be considered as bound by cell surface CD2. Here, this phenomena and fluorescence photobleaching recovery were utilized to determine whether CD2-CD58 bonds are transient in contact areas. Fluorescent CD58 molecules accumulated in the T cell-bilayer interface were completely bleached. The bleached CD58 molecules accumulated in the contact area were rapidly replaced by fluorescent CD58 that diffused into the contact area from adjacent bilayer regions outside the contact area. Rapid recovery of the accumulated fluorescence directly demonstrates that the CD2-CD58 bonds are dissociating and that the dissociation leads to partner exchange, rather than rebinding of the same CD2-CD58 pairs. This suggests that the solution off-rate provides an accurate description of CD2-CD58 interaction in contact areas. Accumulated fluorescent IgG in contacts between K562 cells expressing low affinity Fc receptors and planar bilayers with fluorescent IgG bound to hapten-derivitized phospholipids displayed slower recovery than CD58 by a factor of 10. This suggests that the Fc receptor-IgG interaction has a longer lifetime than the CD2-CD58 interaction. These findings have implications for the mechanism of signaling by CD2 and the mechanism of cell detachment from large numbers of transient interactions.
T细胞糖蛋白CD2与其配体CD58的相互作用对于T细胞与抗原呈递细胞及靶细胞的相互作用至关重要。这种结合相互作用亲和力较低，在溶液中的解离速率较快（>5 s-1）。然而，溶液测量可能无法准确预测分子在黏附接触区域的行为。表达CD2的T细胞与含有纯化且经荧光标记的CD58的玻璃支撑平面双层膜之间的相互作用会导致CD58（荧光）在细胞/双层膜接触区域积累。接触区域内积累的CD58分子超过接触区域外双层膜中CD58密度的部分可被视为与细胞表面CD2结合。在此，利用这一现象和荧光光漂白恢复技术来确定CD2-CD58键在接触区域是否短暂。积累在T细胞-双层膜界面的荧光CD58分子被完全漂白。接触区域积累的漂白CD58分子迅速被从接触区域外相邻双层膜区域扩散进入接触区域的荧光CD58分子所取代。积累荧光的快速恢复直接表明CD2-CD58键正在解离，且这种解离导致伙伴交换，而非相同CD2-CD58对的重新结合。这表明溶液中的解离速率准确描述了接触区域内CD2-CD58的相互作用。在表达低亲和力Fc受体的K562细胞与结合有荧光IgG至半抗原衍生化磷脂的平面双层膜之间的接触中积累的荧光IgG显示出比CD58慢10倍的恢复速度。这表明Fc受体-IgG相互作用的寿命比CD2-CD58相互作用更长。这些发现对CD2的信号传导机制以及细胞从大量短暂相互作用中脱离的机制具有启示意义。
