Binding mechanisms of PEGylated ligands reveal multiple effects of the PEG scaffold
Binding mechanisms of PEGylated ligands reveal multiple effects of the PEG scaffold
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DOI：
10.1021/bi702094j
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发表时间：
2008-01-22
期刊：
影响因子：
2.9
通讯作者：
Goldstein, Byron
中科院分区：
文献类型：
--
作者：
Das, Raibatak;Baird, Emily;Goldstein, Byron
由聚乙二醇（PEG）组成的一系列合成配体，在一端或两端用半抗原2，4-二硝基苯基（DNP）封端，先前显示是由多价抗原刺激的RBL肥大细胞中细胞活化的有效抑制剂[Baird，E. J.，Holowka，D.，科茨，G. W.，和Baird，B.（2003）Biochemistry 42，12739-12748]。在这项研究中，我们系统地研究了PEG支架长度增加对这些单价和二价配体与溶液中抗DNP IgE结合的影响。我们的分析揭示了两个单价配体结合到相同的受体之间的积极有利的相互作用的证据，当PEG分子量超过类似5 kDa。此外，对于具有高得多的分子量（> 10 kDa PEG）的配体，单个配体的结合显然导致大体积PEG支架对第二结合位点的空间排阻。这些结果进一步证实了数据从一个替代的基于荧光的测定，我们开发了定量的能力，这些配体取代一个小的半抗原结合IgE。这种新的检测监测一个小的，受体结合的半抗原的竞争性单价配体的位移，从而定量的竞争性抑制提供了一个单价配体。我们还表明，对于二价配体，抑制能力与IgE形成有效的分子内交联的能力相关。
A series of synthetic ligands consisting of poly(ethylene glycol) (PEG), capped on one or both ends with the hapten 2,4-dinitrophenyl (DNP), were previously shown to be potent inhibitors of cellular activation in RBL mast cells stimulated by a multivalent antigen [Baird, E. J., Holowka, D., Coates, G. W., and Baird, B. (2003) Biochemistry 42, 12739-12748]. In this study, we systematically investigated the effect of increasing length of the PEG scaffold on the binding of these monovalent and bivalent ligands to anti-DNP IgE in solution. Our analysis reveals evidence for an energetically favorable interaction between two monovalent ligands bound to the same receptor, when the PEG molecular mass exceeds similar to 5 kDa. Additionally, for ligands with much higher molecular masses (> 10 kDa PEG), the binding of a single ligand apparently leads to a steric exclusion of the second binding site by the bulky PEG scaffold. These results are further corroborated by data from an alternate fluorescence-based assay that we developed to quantify the capacity of these ligands to displace a small hapten bound to IgE. This new assay monitors the displacement of a small, receptor-bound hapten by a competitive monovalent ligand and thus quantifies the competitive inhibition offered by a monovalent ligand. We also show that, for bivalent ligands, inhibitory capacity is correlated with the capacity to form effective intramolecular cross-links with IgE.