Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin
Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin
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DOI：
10.1074/jbc.271.6.3255
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发表时间：
1996-02-09
影响因子：
4.8
通讯作者：
McEver, RP
中科院分区：
文献类型：
--
作者：
Li, FG;Wilkins, PP;McEver, RP
P-selectin glycoprotein ligand-1（PSGL-1）是人白细胞上P-和E-选择素的粘蛋白样配体。PSGL-1需要唾液酸化、岩藻糖基化的O-连接聚糖和酪氨酸硫酸盐来结合P-选择素。关于PSGL-1结合E-选择素所需的决定簇知之甚少。为了进一步确定PSGL-1结合P-和E-选择素所需的修饰，我们用PSGL-1和特异性糖基转移酶的cDNA转染中国仓鼠卵巢（CHO）细胞。CHO细胞仅合成核心1 O-连接聚糖（Gal β 1-3Gal-NAc α 1-Ser/Thr）;它们缺乏核心2 O-连接聚糖（Gal β 1-3（Gal β 1-4GlcNAc β 1-6）GalNAc α 1- Ser/Thr），因为它们不表达核心2 β 1-6-N-乙酰葡糖胺转移酶（C2 GnT），CHO细胞也缺乏α 1-3岩藻糖基转移酶活性。在转染的CHO细胞上表达的PSGL-1仅在与C2 GnT和α 1-3岩藻糖基转移酶（Fuc-TIII、Fuc-TIV或Fuc-TVII）共表达时才结合P-和E-选择素。来自与C2 GnT共表达的PSGL-1的β-消除的O-连接聚糖的色谱法证实了核心2结构的合成。在CHO细胞中表达的PSGL-1上的酪氨酸残基被硫酸化。酪氨酸硫酸化的共有序列内的三个酪氨酸的苯丙氨酸替换消除了与P-选择素的结合，但不消除与E-选择素的结合。这些结果表明，PSGL-1需要唾液酸化和岩藻糖基化的核心2 O-连接聚糖以结合P-选择素和E-选择素，PSGL-1还需要酪氨酸硫酸盐以结合P-选择素，但不结合E-选择素。
P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Gal beta 1-3Gal-NAc alpha 1-Ser/Thr); they lack core 2 O-linked glycans (Gal beta 1-3(Gal beta 1-4GlcNAc beta 1-6)GalNAc alpha 1- Ser/Thr) because they do not express the core 2 beta 1-6-N-acetylglucosaminyltransferase (C2GnT), CHO cells also lack alpha 1-3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha 1-3 fucosyl-transferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin, These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin, PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.