Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy
Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy
复制标题
DOI：
10.1006/abio.2002.5652
复制
发表时间：
2002-06-15
影响因子：
2.9
通讯作者：
Kiessling, LL
中科院分区：
文献类型：
--
作者：
Gestwicki, JE;Cairo, CW;Kiessling, LL
细胞表面多价配体，如蛋白聚糖和粘蛋白，通常由单个附着点拴系。然而，在体外，由于多价配体的异质性，很难在单个位点上识别多价配体。此外，多价配体通常缺乏具有与配体中的其它官能团正交的反应性的单个基团。多价配体-受体相互作用的生物物理分析将受益于多价配体的均匀固定化策略的可用性。为此，我们报告的设计和合成的多价配体，具有一个单一的终端正交官能团，我们证明，这种材料可以选择性地固定到一个适合表面等离子体共振（SPR）实验的表面上。我们产生的聚合物配体显示多个拷贝的3，6-二硫代半乳糖，它可以结合细胞粘附分子P-和L-选择素。使用SPR测量，我们发现，表面显示我们的多价配体结合特异性P-和L-选择素。P-和L-选择素对展示多价配体的表面的亲和力比对用相应的单价配体修饰的表面的亲和力好五至六倍。除了结合可溶性蛋白质外，带有固定化聚合物的表面与展示L-选择素的细胞结合。通过荧光显微镜观察粘附细胞来确认细胞结合。总之，我们的研究结果表明，合成表面可以通过选择性固定多价配体，这些表面能够结合可溶性和细胞表面相关的受体具有高亲和力。(C)2002 Elsevier Science（美国）。
Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity. (C) 2002 Elsevier Science (USA).