Impact of Epidermal Growth Factor Tethering Strategy on Cellular Response
Impact of Epidermal Growth Factor Tethering Strategy on Cellular Response
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DOI：
10.1021/bc1002604
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发表时间：
2010-12-01
影响因子：
4.7
通讯作者：
De Crescenzo, Gregory
中科院分区：
文献类型：
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作者：
Liberelle, Benoit;Boucher, Cyril;De Crescenzo, Gregory
为了评估各种表皮生长因子（EGF）移植策略对细胞表面受体活化和细胞粘附的影响，我们通过将羧甲基化葡聚糖（CMD）均匀接枝在氨基涂覆的玻璃基底上来产生低污染表面。通过防止非特异性细胞粘附，同时提供反应基团，促进随后的蛋白质移植，CMD允许实现特定的细胞/拴系EGF相互作用，因此得出明确的结论，各种EGF移植策略。我们在这里证明，A-431细胞对固定化EGF的反应高度依赖于被拴系的标记蛋白的生物活性，其正确的方向，和其表面密度。在我们测试的所有方法中，通过全新设计的卷曲螺旋捕获系统定向拴系完全生物活性的EGF被证明是最有效的。也就是说，它导致最强烈和持续的EGF受体磷酸化以及强大的A-431细胞粘附，后者与在没有CMD的情况下用氨基涂覆的表面观察到的情况相当。
In an effort to evaluate the impact of various epidermal growth factor (EGF) grafting strategies upon cell surface receptor activation and cell adhesion, we generated low-fouling surfaces by homogeneously grafting carboxy methylated dextran (CMD) on amino-coated glass substrate. By preventing nonspecific cell adhesion while providing reactive groups facilitating subsequent protein grafting, CMD allowed achieving specific cell/tethered EGF interactions and therefore deriving unambiguous conclusions about various EGF grafting strategies. We demonstrate here that A-431 cell response to immobilized EGF is highly dependent on the bioactivity of the tagged protein being tethered, its proper orientation, and its surface density. Among all the approaches we tested, the oriented tethering of fully bioactive EGF via a de novo-designed coiled-coil capture system was shown to be the most efficient. That is, it led to the most intense and sustained phosphorylation of EGF receptors as well as to strong A-431 cell adhesion, the latter being comparable to that observed with amino-coated surfaces in the absence of CMD.