Matyas G R, Evers D C, Radinsky R, Morré D J
Exp Cell Res. 1986 Feb;162(2):296-318. doi: 10.1016/0014-4827(86)90336-8.
Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (GT1b congruent to GD1b congruent to GD1a greater than GM1 much greater than GM2 congruent to GD3 congruent to GM3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367). Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides GM1, GD1a or GT1b bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent Kd for binding to mixed rat liver gangliosides of 7.8 X 10(-9) M was determined. This value compared favorably with the apparent Kd for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 X 10(-9) M for fibronectin modified on the tyrosine residue, or 6.4 X 10(-9) M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochem biophys acta 550 (1979) 92), fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less [125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.
使用几种不同的体外模型直接测试了纤连蛋白与神经节苷脂的结合。采用酶联免疫吸附测定法（ELISA），将神经节苷脂固定在聚苯乙烯管上，并通过偶联二抗的碱性磷酸酶活性估计纤连蛋白的相对结合情况。在临界神经节苷脂浓度以上，神经节苷脂结合纤连蛋白（GT1b等同于GD1b等同于GD1a大于GM1远大于GM2等同于GD3等同于GM3）的效率顺序与它们在细胞附着试验中竞争纤连蛋白细胞结合位点的顺序大致相同（克莱曼等人，《美国国家科学院院刊》76（1979年）3367）。或者，这些相同的神经节苷脂也与固定化的纤连蛋白结合。用神经节苷脂GM1、GD1a或GT1b包被的大鼠红细胞比未添加神经节苷脂的红细胞结合更多的纤连蛋白。使用赖氨酸残基被放射性碘化的纤连蛋白，测定其与混合大鼠肝脏神经节苷脂结合的表观解离常数（Kd）为7.8×10⁻⁹M。该值与纤连蛋白与大鼠肝脏分离的质膜结合的表观Kd值相比有利，酪氨酸残基修饰的纤连蛋白的表观Kd值为3.7×10⁻⁹M，赖氨酸残基修饰的纤连蛋白的表观Kd值为6.4×10⁻⁹M。如格林内尔和明特之前所示（《生物化学与生物物理学报》550（1979年）92），酪氨酸残基修饰的纤连蛋白不促进CHO细胞的铺展和附着。然而，它确实能与细胞结合。相比之下，赖氨酸修饰的纤连蛋白既能与细胞结合，又能促进细胞附着。从肝肿瘤中分离的质膜，其中结合纤连蛋白的较高神经节苷脂已被耗尽，与对照肝脏的质膜相比，结合的[¹²⁵I]纤连蛋白减少了43 - 75%。这些发现与纤连蛋白与神经节苷脂的结合一致，包括在大鼠肿瘤发生过程中从细胞表面耗尽的相同神经节苷脂。
