Handa K, Stroud M R, Hakomori S
Pacific Northwest Research Foundation, 720 Broadway, Seattle, Washington 98122, USA.
Biochemistry. 1997 Oct 14;36(41):12412-20. doi: 10.1021/bi971181t.
The majority of E- and P-selectin ligands in leukocytes and myelocytic or monocytic leukemia cells are carried by transmembrane glycoproteins having a tandem repeat mucin-like domain through which O-linked carbohydrate ligands are carried. However, determination of structure and adhesive function of carbohydrates in glycoproteins is extremely difficult because of the extensive structural heterogeneity and the scarcity of material for functional analysis. We have overcome this difficulty through use of poly-LacNAc gangliosides isolated from a large quantity of ( approximately 1.2 L packed) HL60 cells [Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., & Reinhold, V. N. (1996) Biochemistry 35, 758-769, 770-778]. We identified two major types of poly-LacNAc gangliosides without the sialosyl-Lex epitope as being capable of binding to E-selectin: (i) those having a single alpha1-->3 fucosylation at internal GlcNAcs but not at the penultimate GlcNAc and (ii) those having double alpha1-->3 fucosylation at internal GlcNAcs, excluding the penultimate GlcNAc. Gangliosides from group i above did not show any adhesion under static conditions, but showed strong adhesion under dynamic flow conditions. Gangliosides from group ii above showed adhesion under both static and dynamic conditions, as did sialosyl-Lex (SLex)-containing structures in previous studies. However, SLex-containing poly-LacNAc gangliosides are virtually absent or present in only trace quantities in leukocytes and HL60 cells. Poly-LacNAc gangliosides from groups i and ii above, lacking SLex structure, are the major membrane components of leukocytes and HL60 cells. These carbohydrates, bound to lipid or to protein, may therefore be the physiological epitope for E-selectin-dependent binding of these cells, particularly under dynamic flow conditions.
白细胞以及髓细胞性或单核细胞性白血病细胞中的大多数E-选择素和P-选择素配体由具有串联重复粘蛋白样结构域的跨膜糖蛋白携带，O-连接的碳水化合物配体通过该结构域携带。然而，由于糖蛋白中碳水化合物的结构高度异质性以及用于功能分析的材料稀缺，确定其结构和粘附功能极其困难。我们通过使用从大量（约1.2升 packed）HL60细胞中分离出的聚乳糖胺神经节苷脂克服了这一困难[斯特劳德，M.R.，汉达，K.，萨利安，M.E.K.，伊藤，K.，勒弗里，S.B.，哈科莫里，S.，莱因霍尔德，B.B.，&莱因霍尔德，V.N.（1996年）《生物化学》35卷，758 - 769页，770 - 778页]。我们鉴定出两种主要类型的不含唾液酸化路易斯表位的聚乳糖胺神经节苷脂能够与E-选择素结合：（i）那些在内侧N-乙酰葡糖胺处有单个α1→3岩藻糖基化但在倒数第二个N-乙酰葡糖胺处没有的；（ii）那些在内侧N-乙酰葡糖胺处有双重α1→3岩藻糖基化，不包括倒数第二个N-乙酰葡糖胺。上述i组的神经节苷脂在静态条件下不显示任何粘附，但在动态流动条件下显示出强粘附。上述ii组的神经节苷脂在静态和动态条件下均显示出粘附，先前研究中含唾液酸化路易斯（SLex）的结构也是如此。然而，含SLex的聚乳糖胺神经节苷脂在白细胞和HL60细胞中实际上不存在或仅以痕量存在。上述i组和ii组不含SLex结构的聚乳糖胺神经节苷脂是白细胞和HL60细胞的主要膜成分。因此，这些与脂质或蛋白质结合的碳水化合物可能是这些细胞依赖E-选择素结合的生理表位，特别是在动态流动条件下。
