Xu Jin, Cai Jun, Suresh M, Peek Simon F, Darien Benjamin J
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706-1102, USA.
Vet Immunol Immunopathol. 2009 Sep 15;131(1-2):33-43. doi: 10.1016/j.vetimm.2009.03.015. Epub 2009 Mar 31.
Equine PSGL-1 (ePSGL-1) is widely expressed on equine PBMC as a homodimer with sialylation (sLeX) modifications that contribute to P-selectin binding affinity. To investigate the role of other potential post-translational modifications required for high-affinity P-selectin binding, ePSGL-1 was transfected into CHO cells expressing equine FucT-VII and/or C2GnT. P-selectin-IgG chimera binding by ePSGL-1 transfected into CHO cells only occurred when both FucT-VII and C2GnT were expressed, establishing that fucosylation and core-2 branching are required as post-translational modifications for high-affinity P-selectin binding. However, enzymatic removal of N-glycans or site and/or point-mutation preventing N-glycan addition did not inhibit P-selectin binding, indicating that N-glycosylation is not required. Taken together, we hypothesized that sialylation, fucosylation, or core-2 branching must occur on O-glycans. The presence of numerous serine/threonine residues in the ePSGL-1 extracellular domain suggests several potential O-glycans attachment sites. P-selectin binding was also susceptible to OSGP cleavage, providing evidence for the existence of clustered, sialyated O-glycans on ePSGL-1. Because OSGP eliminated ePSGL-1 precipitation the P-selectin binding domain of ePSGL-1 must contain clustered, sialyated, fucosylated, and core-2 branched O-glycans. Using point-mutation deletion techniques, the binding domain was determined to reside between residues 48 and 100 of ePSGL-1. Sulfation, a critical modification for human PSGL-1 binding to P-selectin, was not necessary for equine P-selectin binding, while dimerization of ePSGL-1 was critical. These species-specific features of equine PSGL-1 provide new information that advances our understanding of high-affinity P-selectin binding mediated mononuclear cell trafficking.
马PSGL-1（ePSGL-1）作为一种具有唾液酸化（sLeX）修饰的同型二聚体在马外周血单核细胞（PBMC）上广泛表达，这些修饰有助于P-选择素结合亲和力。为了研究高亲和力P-选择素结合所需的其他潜在翻译后修饰的作用，将ePSGL-1转染到表达马FucT-VII和/或C2GnT的CHO细胞中。只有当FucT-VII和C2GnT都表达时，转染到CHO细胞中的ePSGL-1才能与P-选择素-IgG嵌合体结合，这表明岩藻糖基化和核心2分支作为高亲和力P-选择素结合的翻译后修饰是必需的。然而，酶促去除N-聚糖或阻止N-聚糖添加的位点和/或点突变并不抑制P-选择素结合，表明不需要N-糖基化。综上所述，我们推测唾液酸化、岩藻糖基化或核心2分支必须发生在O-聚糖上。ePSGL-1细胞外结构域中存在大量丝氨酸/苏氨酸残基，提示了几个潜在的O-聚糖附着位点。P-选择素结合也易受OSGP切割的影响，这为ePSGL-1上存在聚集的、唾液酸化的O-聚糖提供了证据。因为OSGP消除了ePSGL-1沉淀，所以ePSGL-1的P-选择素结合结构域必须包含聚集的、唾液酸化的、岩藻糖基化的和核心2分支的O-聚糖。使用点突变缺失技术，确定结合结构域位于ePSGL-1的第48至100个残基之间。硫酸化是人类PSGL-1与P-选择素结合的关键修饰，但对马P-选择素结合不是必需的，而ePSGL-1的二聚化是关键的。马PSGL-1的这些物种特异性特征提供了新信息，推进了我们对高亲和力P-选择素结合介导的单核细胞运输的理解。
