Smith McRae J, Smith Bryan R E, Lawrence Michael B, Snapp Karen R
Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22903, USA.
J Biol Chem. 2004 May 21;279(21):21984-91. doi: 10.1074/jbc.M402731200. Epub 2004 Mar 16.
Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.
白细胞P-选择素糖蛋白配体-1（PSGL-1）以同二聚体形式表达，并通过与内皮P-选择素相互作用介导白细胞滚动。先前的研究表明，PSGL-1必须由特定的糖基转移酶进行适当修饰，这些酶包括α1,3-岩藻糖基转移酶-VII、核心2β1-6-N-乙酰葡糖胺基转移酶（C2GlcNAcT-I）、一种或多种α2,3-唾液酸转移酶和一种酪氨酸硫酸转移酶。此外，PSGL-1通过其唯一的细胞外半胱氨酸（Cys(320)）进行二聚化对于在剪切条件下在P-选择素上滚动至关重要。在本报告中，我们测量了C2GlcNAcT-I糖基化和PSGL-1二聚化对转染细胞系在纯化的P-选择素上进行系留和滚动过程中形成的粘附键的贡献。与表达单体PSGL-1（称为C320A）的细胞相比，在流动条件下与P-选择素的系留随着二聚化而增加。表达单体PSGL-1的转染子所表现出的滚动缺陷（细胞积累减少、PSGL-1/P-选择素键强度和系留率降低、速度和跳跃距离增加）可以通过增加底物P-选择素位点密度以及在C320A转染子中过表达C2GlcNAcT-I来克服。从用PSGL-1、C320A和/或C2GlcNAcT-I cDNA转染的细胞系中分离出两种分子量变体的PSGL-1，这些电泳迁移率的差异似乎与C2GlcNAcT-I表达相关。与具有高水平C2GlcNAcT-I和高分子量PSGL-1的C320A细胞相比，表达低分子量PSGL-1的C320A转染子具有较低的C2GlcNAcT-I水平（通过对核心2特异性连接抗体CHO-131的反应性测量），并且在P-选择素上的滚动受损（无论位点密度如何）。C2GlcNAcT-I糖基化和PSGL-1二聚化均增加了在流动条件下与P-选择素的系留率，而C2GlcNAcT-I水平主要影响系留键强度。
