Snapp K R, Craig R, Herron M, Nelson R D, Stoolman L M, Kansas G S
Department of Microbiology/Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
J Cell Biol. 1998 Jul 13;142(1):263-70. doi: 10.1083/jcb.142.1.263.
Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with alpha1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.
在内皮细胞和活化血小板上表达的P-选择素与其白细胞配体（一种称为P-选择素糖蛋白配体-1，即PSGL-1的同二聚体）之间的相互作用，介导了炎症反应期间最早的黏附事件。为了研究PSGL-1的二聚化对于与P-选择素的功能相互作用是否至关重要，生成了一种PSGL-1的突变形式，其中保守的膜近端半胱氨酸突变为丙氨酸（命名为C320A）。在从稳定转染细胞中分离出的C320A PSGL-1突变体的变性和天然条件下进行的蛋白质免疫印迹分析显示，仅表达单体形式的PSGL-1。与共转染α1-3岩藻糖基转移酶-VII（FucT-VII）加PSGL-1的细胞相反，表达FucT-VII加C320A的K562细胞在低剪切力黏附试验中未能结合转染了P-选择素的COS细胞，也未能在生理流动条件下在转染了P-选择素的CHO细胞上滚动。此外，C320A转染子未能结合嵌合P-选择素融合蛋白。PSGL-1和C320A均均匀分布在转染的K562细胞表面。因此，PSGL-1通过单个保守的细胞外半胱氨酸进行二聚化对于P-选择素的功能识别至关重要。
