Department of Chemistry, and Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA.
J Am Chem Soc. 2012 Jun 13;134(23):9549-52. doi: 10.1021/ja301694s. Epub 2012 May 30.
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.
凝集素家族的糖结合蛋白被认为通过将细胞表面糖蛋白和糖脂寡聚化为更高阶的聚集物来介导许多细胞过程。这一假设反映了凝集素自身的已知寡聚状态及其在体外与多价配体的结合特性，但缺乏它们在细胞表面交联配体的能力的直接证据。凝集素-配体相互作用的基础研究中的一个主要挑战是，它们的天然配体包含一组异质的糖缀合物，这些糖缀合物具有相关的聚糖结构，但潜在的支架却大不相同。因此，没有明显的方法可以选择性地监测活细胞表面上天然凝集素配体的行为。在这里，我们描述了一种用于探测培养细胞上糖缀合物的凝集素诱导的多聚化的方法。我们使用 RAFT 聚合，沿着主链合成了具有凝集素结合聚糖的、定义明确的糖聚合物（GP），在一端有一个脂质基团，另一端有一个荧光团。插入活细胞膜后，在存在和不存在凝集素-1 的情况下测量了 GP 的荧光寿命和扩散时间。我们观察到了直接证据，证明了凝集素-1 介导的工程细胞上的延伸交联，这种现象依赖于聚糖结构。该平台为探索“凝集素晶格”假说以及在生理相关环境中定义凝集素配体特异性提供了一种新方法。
