Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity.
Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity.
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发表时间：
1994-04
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通讯作者：
Minghze Zheng;H. Fang;S. Hakomori
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作者：
Minghze Zheng;H. Fang;S. Hakomori
纤连蛋白（FN）介导的细胞粘附主要由α 5 β 1（识别RGD序列）和α 4 β 1（识别FN的CS-1肽序列）整联蛋白受体控制。整合素依赖性细胞与FN的粘附通过在表面膜处的最佳GM 3浓度大大促进（Zheng，M.，方，H.，鹤冈，T.，Tsuji，T.，Sasaki，T.，和Hakomori，S.（1993）J.Biol.Chem.268，2217-2222），并且通过修饰整联蛋白受体的N-糖基化加工来抑制由α 4 β 1（对FN）或α 6 β 1（对层粘连蛋白）介导的细胞粘附（例如Akiyama，S. K.，山田，S。美国，和Yamada，K. M.（1989）J.Biol.Chem.264，18011-18018）。因此，我们研究了N-糖基化在α 5 β 1功能中的具体作用。本研究的主要结果如下。(i)当用内切-N-乙酰葡糖胺糖苷酶F和肽-N4-（N-乙酰葡糖胺酰基）天冬酰胺酰胺酶F（内切-F/PNGase-F）的混合物处理细胞时，K562细胞对FN包被的板的粘附（其仅由α 5 β 1介导）被抑制。(ii)当用endo-F/PNGase-F处理的细胞提取物被整合素亚基特异性抗体沉淀时，K562细胞表面的α 5 β 1受体倾向于解离成α 5和β 1亚基，即α 5亚基优先被抗α 5单克隆抗体ZH 5沉淀，抗β 1单克隆抗体ZH 1优先沉淀β 1亚基。当提取完整的细胞并用ZH 5或ZH 1处理时，α 5和β 1都被共沉淀，表明这两个亚基通常彼此紧密相关。(iii)通过用endo-F/PNGase-F处理脂质体来消除含有α 5 β 1的脂质体（磷脂酰胆碱：胆固醇脂质体，掺入纯化的α 5 β 1）与FN包被的板的粘附。掺入用endo-F/PNGase-F预处理的α 5 β 1的脂质体也不与FN结合。当用endo-F/PNGase-F处理纯化的α 5 β 1受体，然后用ZH 5或ZH 1处理时，α 5或β 1亚基分别沉淀。相反，当用ZH 5或ZH 1直接处理完整纯化的α 5 β 1受体时，两种亚基总是共沉淀。这些发现表明，α 5 β 1整联蛋白受体α和β亚基的N-糖基化对于这些亚基的结合和与FN的最佳结合是必不可少的。
Fibronectin (FN)-mediated cell adhesion is controlled mainly by alpha 5 beta 1 (recognizing the RGD sequence) and alpha 4 beta 1 (recognizing the CS-1 peptide sequence of FN) integrin receptors. Integrin-dependent cell adhesion to FN is greatly promoted by optimal GM3 concentration at the surface membrane (Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-2222), and cell adhesion mediated by alpha 4 beta 1 (to FN) or alpha 6 beta 1 (to laminin) is inhibited by modifying N-glycosylation processing of the integrin receptor (e.g. Akiyama, S. K., Yamada, S. S., and Yamada, K. M. (1989) J. Biol. Chem. 264, 18011-18018). We therefore studied the specific role of N-glycosylation in alpha 5 beta 1 function. Key findings of the present study were as follows. (i) Adhesion of K562 cells to FN-coated plates, which is mediated solely by alpha 5 beta 1, was inhibited when cells were treated with a mixture of endo-N-acetylglucosaminidase F and peptide -N4-(N-acetylglucosaminyl)asparagine amidase F (endo-F/PNGase-F). (ii) The alpha 5 beta 1 receptor at the K562 cell surface tended to dissociate into alpha 5 and beta 1 subunits when an extract of cells treated with endo-F/PNGase-F was precipitated by integrin subunit-specific antibodies, i.e. the alpha 5 subunit was preferentially precipitated by anti-alpha 5 monoclonal antibody ZH5, and the beta 1 subunit was preferentially precipitated by anti-beta 1 monoclonal antibody ZH1. When intact cells were extracted and treated with either ZH5 or ZH1, both alpha 5 and beta 1 were coprecipitated, indicating that the two subunits are normally tightly associated with each other. (iii) Adhesion of alpha 5 beta 1-containing liposomes (phosphatidylcholine:cholesterol liposomes incorporating purified alpha 5 beta 1) to FN-coated plates was abolished by treatment of liposomes with endo-F/PNGase-F. Liposomes incorporating alpha 5 beta 1 pretreated with endo-F/PNGase-F also did not bind to FN. When purified alpha 5 beta 1 receptor was treated with endo-F/PNGase-F followed by ZH5 or ZH1, the alpha 5 or beta 1 subunit was precipitated separately, respectively. In contrast, both subunits were always coprecipitated when intact purified alpha 5 beta 1 receptor was directly treated with ZH5 or ZH1. These findings indicate that N-glycosylation of both the alpha and beta subunits of the alpha 5 beta 1 integrin receptor is essential for association of these subunits and for optimal binding to FN.