Double-modification of lectin using two distinct chemistries for fluorescent ratiometric sensing and imaging saccharides in test tube or in cell.
Double-modification of lectin using two distinct chemistries for fluorescent ratiometric sensing and imaging saccharides in test tube or in cell.
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DOI：
10.1021/ja052731a
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发表时间：
2005-09
影响因子：
15
通讯作者：
Eiji Nakata;Yoichiro Koshi;E. Koga;Y. Katayama;I. Hamachi
中科院分区：
文献类型：
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作者：
Eiji Nakata;Yoichiro Koshi;E. Koga;Y. Katayama;I. Hamachi
使用两种不同的正交化学方法，已成功地进行了两种不同的荧光团的位点选择性掺入到天然存在的蛋白质（凝集素，糖结合蛋白）。通过后光亲和标记修饰，Con A，葡萄糖和甘露糖选择性凝集素，被修改与荧光素在糖结合位点（Tyr 100位点）的附近，和受控的酰化反应提供了在Lys 114的香豆素的位点选择性连接。在这种双重修饰的Con A中，荧光素发射在与相应的糖（例如葡萄糖或甘露糖衍生物）结合时改变，而香豆素发射是恒定的。因此，双重修饰的Con A荧光感测的葡萄糖和甘露糖丰富的葡萄糖在比率的方式，同时保留天然结合的选择性和亲和力，无论双修饰。利用两种不同探针的比率荧光分析法，可以通过工程化的Con A精确地监测糖蛋白的糖修剪过程。此外，双重修饰的Con A不仅可以用于位于细胞表面的葡萄糖的方便的荧光成像，例如MCF-7，一种具有丰富的高甘露糖分支的乳腺癌细胞，而且可以用于HepG 2细胞内葡萄糖浓度的比率荧光传感。这些结果表明，经两种不同化学修饰的半合成凝集素在功能上上级单一修饰的凝集素，因此，它可能在细胞中以及在试管中具有潜在的用途。
The site-selective incorporation of two different fluorophores into a naturally occurring protein (lectin, a sugar-binding protein) has been successfully carried out using two distinct orthogonal chemical methods. By post-photoaffinity labeling modification, Con A, a glucose- and mannose-selective lectin, was modified with fluorescein in the proximity of the sugar binding site (Tyr100 site), and the controlled acylation reaction provided the site-selective attachment of coumarin at Lys114. In this doubly modified Con A, the fluorescein emission changed upon the binding to the corresponding sugars, such as the glucose or mannose derivatives, whereas the coumarin emission was constant. Thus, the doubly modified Con A fluorescently sensed the glucose- and mannose-rich saccharides in a ratiometric manner while retaining the natural binding selectivity and affinity, regardless of the double modification. On the benefit of the ratiometric fluorescent analysis using two distinct probes, the sugar trimming process of a glycoprotein can be precisely monitored by the engineered Con A. Furthermore, the doubly modified Con A can be used not only for the convenient fluorescent imaging of saccharides localized on a cell surface, such as the MCF-7, a breast cancer cell having rich high-mannose branch, but also for the ratiometric fluorescent sensing of the glucose concentration inside HepG2 cells. These results demonstrated that the semisynthetic lectin modified doubly by two distinct chemistries is superior to the singly modified one in function, and thus, it may be potentially useful in cell, as well as in test tube.