Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hyaluronan.
Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hyaluronan.
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DOI：
10.1083/jcb.140.2.431
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发表时间：
1998-01-26
期刊：
影响因子：
--
通讯作者：
Stamenkovic I
中科院分区：
文献类型：
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作者：
Skelton TP;Zeng C;Nocks A;Stamenkovic I
糖基化参与了CD44介导的透明质酸(HA)细胞结合的调节。然而，无论是N-和O-连接的糖链还是改变CD44与HA亲和力的寡糖结构的相对贡献都还没有阐明。为了确定CD44寡糖组成的选择性改变对CD44与HA亲和力的影响，我们开发了一种基于亲和毛细管电泳法(ACE)的新策略。CD44-免疫球蛋白融合蛋白在突变型CHO细胞系LDL-D中过量表达，该突变株在N-和O-连接的寡糖合成方面均存在可逆缺陷。利用该细胞系、一组重组糖苷酶和代谢性糖苷酶抑制剂，产生了具有明确寡糖结构的CD44糖型，并通过ACE检测了其与HA的亲和力。由于低密度脂蛋白-D细胞表达内源性细胞表面CD44，因此可以比较任何糖基化改变对细胞表面和可溶性CD44结合HA能力的影响。研究发现四种不同的寡糖结构对CD44介导的HA结合有影响：(A)N-连接的寡糖上的末端α-2，3-连接的唾液酸抑制结合；(B)N-连接的N-乙酰氨基葡萄糖残基增强结合；(C)O-连接的糖链在N-脱糖基化的CD44上增强结合；(D)N-乙酰半乳糖胺掺入非N-连接的糖链可增强细胞表面CD44与HA的结合。前三种结构诱导对HA的固有CD44亲和力发生30倍的变化(Kd=5 to>；150μM)。第四种方法增强了CD44介导的细胞HA亲和力，而不改变可溶性CD44固有的HA亲和力。
Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44–immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal α2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non–N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (K d = 5 to >150 μM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.