Ghoshal Pushpankur, Rajendran Mythilypriya, Odo Nadine, Ikuta Tohru
Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia, United States of America.
PLoS One. 2014 Jun 19;9(6):e99363. doi: 10.1371/journal.pone.0099363. eCollection 2014.
Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.
黏附分子在镰状细胞病（SCD）中多种细胞类型的黏附相互作用中起关键作用。我们之前表明，抗P-选择素适配体可有效抑制细胞与内皮细胞（EC）的黏附，并使SCD小鼠在缺氧应激下存活。为了发现抑制P-选择素的新机制，我们研究了糖基化的作用。P-选择素是一种90 kDa的蛋白质，但在凝胶电泳上发现其迁移为90 kDa和140 kDa的条带。当用肽N-糖苷酶F而非O-糖苷酶消化从EC中分离的P-选择素时，140 kDa的条带消失，90 kDa的条带增强。用衣霉素（一种N-糖基化抑制剂）处理EC，可抑制细胞表面CD62P（P-选择素）的表达以及细胞质中140 kDa形式的表达。这些结果表明，140 kDa的条带是N-糖基化的，糖基化对于EC中P-选择素的细胞表面表达至关重要。刺激EC上P-选择素表达的凝血酶可诱导AKT磷酸化，而衣霉素可抑制AKT磷酸化，这表明AKT信号传导参与了衣霉素介导的P-选择素表达抑制。重要的是，两种结构不同的糖基化抑制剂可显著抑制凝血酶或缺氧诱导的镰状红细胞（sRBC）和白细胞与EC的黏附；其水平与在体内最强烈抑制细胞黏附的P-选择素单克隆抗体相当。在EC中使用短发夹RNA对P-选择素进行敲低研究可抑制sRBC黏附，表明P-选择素在sRBC黏附中起合理作用。总之，这些结果表明，EC上P-选择素的表达部分受糖基化机制调节，并且糖基化抑制剂可有效减少sRBC和白细胞与EC的黏附。糖基化抑制剂可能会导致一种抑制SCD中细胞黏附的新疗法。
