Identification of N-terminal residues on P-selectin glycoprotein ligand-1 required for binding to P-selectin
Identification of N-terminal residues on P-selectin glycoprotein ligand-1 required for binding to P-selectin
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DOI：
10.1074/jbc.273.12.7078
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发表时间：
1998-03-20
影响因子：
4.8
通讯作者：
McEver, RP
中科院分区：
文献类型：
--
作者：
Liu, W;Ramachandran, V;McEver, RP
人白细胞上P-选择素的主要高亲和力配体是P-选择素糖蛋白配体-I（PSGL-1）。为了结合P-选择素，PSGL-1必须用酪氨酸硫酸盐和唾液酸化、岩藻糖基化的核心-α 0-聚糖修饰。在全长PSGL-1上进行这些修饰所需的位点尚未确定。成熟PSGL-1的N-末端区域从残基42开始，包括残基46、48和51处的酪氨酸，加上残基44和57处的Thr连接的O-聚糖的潜在位点。将细胞与在PSGL-1上构建唾液酸化和岩藻糖基化的核心-2 O-聚糖所需的糖基转移酶的cDNA共转染。测定转染细胞结合液相P-选择素和支持在流体动力学流动下表达P-选择素的前B细胞的滚动粘附的能力。在两种测定中，用丙氨酸取代Thr-57消除PSGL-1与P-选择素的结合而不影响PSGL-1的硫酸化，而用丝氨酸取代（O-聚糖也可能附着于丝氨酸）不影响结合。通过用丙氨酸取代Thr-57任一侧的两个氨基酸或用丙氨酸取代Thr-44，结合没有改变。用苯丙氨酸取代所有三种酪氨酸显著减少硫酸化并防止与P-选择素结合。然而，其中一个或两个酪氨酸被苯丙氨酸取代的所有构建体都结合P-选择素。这些结果表明全长PSGL-1需要连接到Thr-57的0-聚糖加上其三个成簇的酪氨酸中的任何一个的硫酸化以结合P-选择素。
The major high affinity ligand for P-selectin on human leukocytes is P-selectin glycoprotein ligand-l (PSGL-1), To bind P-selectin, PSGL-1 must be modified with tyrosine sulfate and sialylated, fucosylated, core-a O-glycan(s). The required sites for these modifications on full-length PSGL-1 have not been defined, The N-terminal region of mature PSGL-1, which begins at residue 42, includes tyrosines at residues 46, 48, and 51, plus potential sites for Thr-linked O-glycans at residues 44 and 57, We expressed full-length PSGL-1 constructs with substitutions of these residues in transfected Chinese hamster ovary cells. The cells were co-transfected with cDNAs for the glycosyltransferases required to construct sialylated and fucosylated, core-2 O-glycans on PSGL-1. The transfected cells were assayed for their abilities to bind fluid-phase P-selectin and to support rolling adhesion of pre-B cells expressing P-selectin under hydrodynamic flow, In both assays, substitution of Thr-57 with alanine eliminated binding of PSGL-1 to P-selectin without affecting sulfation of PSGL-1, whereas substitution with serine, to which an O-glycan might also be attached, did not affect binding. Binding was not altered by substituting alanines for the two amino acids on either side of Thr-57, or by substituting alanine for Thr-44. Substitution of all three tyrosines with phenylalanines markedly reduced sulfation and prevented binding to P-selectin. However, all constructs in which one or two tyrosines were replaced with phenylalanines bound P-selectin, These results suggest that full-length PSGL-1 requires an O-glycan attached to Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.