Ushiyama S, Laue T M, Moore K L, Erickson H P, McEver R P
W. K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
J Biol Chem. 1993 Jul 15;268(20):15229-37.
P-selectin is an adhesion receptor for leukocytes on thrombin-activated platelets and endothelial cells. It contains a NH2-terminal carbohydrate-recognition domain, an epidermal growth factor motif, nine consensus repeats, a transmembrane domain, and a cytoplasmic tail. We expressed two soluble forms of P-selectin, one truncated after the ninth repeat (tPS) and the other lacking the transmembrane domain due to alternative RNA splicing (asPS). When visualized by electron microscopy, each was a monomeric rod-like structure with a globular domain at one end, whereas membrane P-selectin (mPS) from platelets formed rosettes with the globular domains facing outward. Sedimentation velocity and equilibrium studies confirmed that tPS and asPS were asymmetric monomers, whereas mPS was oligomeric. HL-60 cells adhered to immobilized tPS and asPS, although less efficiently than to mPS. 125I-Labeled tPS and asPS bound to approximately 25,000 sites/neutrophil and approximately 36,000 sites/HL-60 cell with an apparent Kd of 70 nM. Treatment of HL-60 cells with O-sialoglycoprotease eliminated the binding sites for asPS. We conclude that 1) P-selectin is a rigid, asymmetric protein; 2) monomeric soluble P-selectin binds to high affinity ligands with sialylated O-linked oligosaccharides on leukocytes; and 3) oligomerization of mPS enhances its avidity for leukocytes.
P-选择素是凝血酶激活的血小板和内皮细胞上白细胞的黏附受体。它包含一个氨基末端碳水化合物识别结构域、一个表皮生长因子基序、九个共有重复序列、一个跨膜结构域和一个细胞质尾巴。我们表达了两种可溶性形式的P-选择素，一种在第九个重复序列后被截断（tPS），另一种由于可变RNA剪接而缺少跨膜结构域（asPS）。通过电子显微镜观察，每种都是一端带有球状结构域的单体杆状结构，而来自血小板的膜P-选择素（mPS）形成了球状结构域朝外的玫瑰花结。沉降速度和平衡研究证实tPS和asPS是不对称单体，而mPS是寡聚体。HL-60细胞黏附于固定化的tPS和asPS，尽管效率低于黏附于mPS。125I标记的tPS和asPS以约70 nM的表观解离常数与约25,000个位点/中性粒细胞和约36,000个位点/HL-60细胞结合。用O-唾液酸糖蛋白酶处理HL-60细胞消除了asPS的结合位点。我们得出结论：1）P-选择素是一种刚性的不对称蛋白质；2）单体可溶性P-选择素与白细胞上带有唾液酸化O-连接寡糖的高亲和力配体结合；3）mPS的寡聚化增强了其对白细胞的亲和力。
