Investigating cell surface galectin-mediated cross-linking on glycoengineered cells.
Investigating cell surface galectin-mediated cross-linking on glycoengineered cells.
复制标题
DOI：
10.1021/ja301694s
复制
发表时间：
2012-06-13
影响因子：
15
通讯作者：
Bertozzi, Carolyn R.
中科院分区：
文献类型：
--
作者：
Belardi, Brian;O'Donoghue, Geoff P.;Smith, Adam W.;Groves, Jay T.;Bertozzi, Carolyn R.
半乳糖凝集素家族的糖链结合蛋白被认为通过将细胞表面糖蛋白和糖脂寡聚为高阶聚集体来调节许多细胞过程。这一假说反映了Galectins本身的已知低聚状态以及它们与体外多价配体的结合特性，但缺乏直接证据表明它们能够在细胞表面交联配体。半乳糖凝集素-配体相互作用的基础研究中的一个主要挑战是，它们的天然配体包括一组不同的糖共轭化合物，这些糖共轭化合物共享相关的糖链结构，但不同的底层支架。因此，没有明显的手段来选择性地监测活细胞表面天然半乳糖凝集素配体的行为。在这里，我们描述了一种方法，用于探测半乳糖凝集素在培养细胞上诱导的糖共轭多聚体。通过RAFT聚合，我们合成了功能明确的糖共聚物(GP)，主链上有半乳糖凝集素结合的多糖，一端是脂基，另一端是荧光团。将GPs插入活细胞膜后，在Galectin-1存在和不存在的情况下，测量GPs的荧光寿命和扩散时间。我们观察到了半乳糖凝集素-1在工程细胞上介导的延长交联度的直接证据，这一现象依赖于多糖结构。这一平台提供了一种新的方法来探索“Galectin晶格”假说，并在生理相关的背景下定义Galectin配体的特异性。
The galectin family of glycan-binding proteins is thought to mediate many cellular processes by oligomerizing cell surface glycoproteins and glycolipids into higher-order aggregates. This hypothesis reflects the known oligomeric states of the galectins themselves and their binding properties with multivalent ligands in vitro, but direct evidence of their ability to cross-link ligands on a cell surface is lacking. A major challenge in fundamental studies of galectin–ligand interactions is that their natural ligands comprise a heterogeneous collection of glycoconjugates that share related glycan structures but disparate underlying scaffolds. Consequently, there is no obvious means to selectively monitor the behaviors of natural galectin ligands on live cell surfaces. Here we describe an approach for probing the galectin-induced multimerization of glycoconjugates on cultured cells. Using RAFT polymerization, we synthesized well-defined glycopolymers (GPs) functionalized with galectin-binding glycans along the backbone, a lipid group on one end and a fluorophore on the other. After insertion into live cell membranes, the GPs’ fluorescence lifetime and diffusion time were measured in the presence and absence of galectin-1. We observed direct evidence for galectin-1-mediated extended cross-linking on the engineered cells, a phenomenon that was dependent on glycan structure. This platform offers a new approach to exploring the “galectin lattice” hypothesis and to defining galectin ligand specificity in a physiologically relevant context.
登录
查看更多内容
影响因子：
64.5
作者：
KINGSLEY, DM;KOZARSKY, KF;KRIEGER, M
通讯作者：
KRIEGER, M
影响因子：
56.9
作者：
Partridge, EA;Le Roy, C;Dennis, JW
通讯作者：
Dennis, JW
影响因子：
2.9
作者：
Di Lella, Santiago;Sundblad, Victoria;Cerliani, Juan P.;Guardia, Carlos M.;Estrin, Dario A.;Vasta, Gerardo R.;Rabinovich, Gabriel A.
通讯作者：
Rabinovich, Gabriel A.
影响因子：
15
作者：
Rawat, Manish;Gama, Cristal I.;Hsieh-Wilson, Linda C.
通讯作者：
Hsieh-Wilson, Linda C.
DOI：
10.1073/pnas.0807207106
发表时间：
2009-02-24
影响因子：
11.1
作者：
Courtney, Adam H.;Puffer, Erik B.;Kiessling, Laura L.
通讯作者：
Kiessling, Laura L.