Expressing murine beta 1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferase-dependent phenotype.
Expressing murine beta 1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferase-dependent phenotype.
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DOI：
10.1016/s0021-9258(18)46887-1
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发表时间：
1994-11
期刊：
影响因子：
--
通讯作者：
T. Nguyen;D. A. Hinton;B. Shur
中科院分区：
文献类型：
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作者：
T. Nguyen;D. A. Hinton;B. Shur
β 1，4-半乳糖基转移酶传统上被视为高尔基复合体的生物合成组分，但半乳糖基转移酶的一部分也在细胞表面上表达，在那里它被认为是作为细胞外寡糖配体的受体发挥作用。虽然来自各种研究的结果与半乳糖基转移酶的细胞粘附功能一致，但表面半乳糖基转移酶功能的最严格测试是在通常表达可忽略水平的表面半乳糖基转移酶的细胞中产生表面半乳糖基转移酶依赖性表型。与以前的报道一致，发现人HeLa细胞在其表面上表达低水平的半乳糖基转移酶，因此，用编码鼠半乳糖基转移酶的cDNA稳定转染。鼠半乳糖基转移酶内的假定高尔基复合体和细胞表面上的表达，作为测定的酶活性和抗血清提出了对细菌表达的鼠酶。HeLa细胞转染子比对照转染子更强烈地粘附于其细胞外基质，如培养物中更平坦的形态和铺板后更快速的铺展所证明的。相比之下，细胞铺展是低的，并且当在不含半乳糖基转移酶结合位点的细胞外基质上铺板时，在所有细胞类型中是相似的。针对重组鼠半乳糖基转移酶的抗体和Fab片段抑制了半乳糖基转移酶转染子的细胞扩散特性的增加，可溶性重组半乳糖基转移酶和各种半乳糖基转移酶干扰物也是如此。因此，异源半乳糖基转移酶的表达产生表面半乳糖基转移酶依赖性表型，证实其作为细胞粘附分子的功能。
Beta 1,4-Galactosyltransferase is traditionally viewed as a biosynthetic component of the Golgi complex, but a portion of galactosyltransferase is also expressed on the cell surface, where it has been suggested to function as a receptor for extracellular oligosaccharide ligands. Although results from a variety of studies are consistent with a cell adhesion function for galactosyltransferase, the most rigorous test of surface galactosyltransferase function is to produce a surface galactosyltransferase-dependent phenotype in cells that normally express negligible levels of surface galactosyltransferase. In agreement with previous reports, human HeLa cells were found to express low levels of galactosyltransferase on their surface and, therefore, were stably transfected with cDNAs encoding murine galactosyltransferase. Murine galactosyltransferase was expressed both within the presumed Golgi complex and on the cell surface, as assayed by enzyme activity and with antiserum raised against the bacterially expressed murine enzyme. HeLa cell transfectants adhered more strongly to their extracellular substrates than did control transfectants, as evidenced by a flatter morphology in culture and a more rapid spreading upon plating. In contrast, cell spreading was low and similar among all cell types when plated on extracellular substrates that did not contain binding sites for galactosyltransferase. Antibodies and Fab fragments against recombinant murine galactosyltransferase inhibited the increased cell spreading characteristic of galactosyltransferase transfectants, as did soluble recombinant galactosyltransferase and a variety of galactosyltransferase perturbants. Thus, expression of heterologous galactosyltransferase produces a surface galactosyltransferase-dependent phenotype, confirming its function as a cell adhesion molecule.