Mondal Nandini, Stolfa Gino, Antonopoulos Aristotelis, Zhu Yuqi, Wang Shuen-Shiuan, Buffone Alexander, Atilla-Gokcumen G Ekin, Haslam Stuart M, Dell Anne, Neelamegham Sriram
From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
Arterioscler Thromb Vasc Biol. 2016 Apr;36(4):718-27. doi: 10.1161/ATVBAHA.115.306748. Epub 2016 Feb 11.
Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion.
A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished.
Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.
近期研究表明，人类白细胞上表达的E选择素配体可能与其他物种（尤其是小鼠）的不同。为详细阐述这一点，我们评估了人类髓细胞上表达的糖鞘脂在调节E选择素介导的细胞黏附中的作用。
通过使用慢病毒传递的短发夹RNA或基于CRISPR-Cas9的基因组编辑靶向UDP-葡萄糖神经酰胺葡萄糖基转移酶，创建了一系列经过修饰的人类细胞系和原代中性粒细胞。酶学和质谱分析证实，修饰后的细胞中神经酰胺葡萄糖生物合成减少或消除。糖组学分析表明，UDP-葡萄糖神经酰胺葡萄糖基转移酶的破坏还增加了平分型N聚糖的发生率，并降低了白细胞N聚糖和O聚糖上唾液酸聚糖的整体表达。基于微流控的流动腔研究表明，UDP-葡萄糖神经酰胺葡萄糖基转移酶基因敲除和敲低的细胞在表达E选择素的刺激内皮细胞上的白细胞滚动和牢固黏附均降低了约60%，而不改变细胞对P选择素的黏附。与糖鞘脂支持缓慢滚动并向牢固黏附转变的概念一致，抑制UDP-葡萄糖神经酰胺葡萄糖基转移酶活性导致白细胞频繁脱离事件、跳跃运动以及跨内皮细胞的穿膜运动减少。带有截短的O聚糖和N聚糖的细胞在E选择素上也能维持细胞滚动，尽管它们从自由流体流动中被募集的能力有所下降。
糖鞘脂可能有助于人类髓细胞在流体剪切力作用下与E选择素的黏附，特别是滚动细胞向牢固黏附的转变。
