Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants
Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants
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DOI：
10.1021/bi051144z
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发表时间：
2005-09-20
期刊：
影响因子：
2.9
通讯作者：
Brewer, CF
中科院分区：
文献类型：
--
作者：
Dam, TK;Gabius, HJ;Brewer, CF
我们先前对合成的多价碳水化合物与Man/Glc特异性凝集素伴刀豆球蛋白A（ConA）和大花山龙眼凝集素（DGL）的结合的等温滴定微量热法（ITC）研究显示负结合协同性，这是由于碳水化合物配体而不是蛋白质[Dam，T. K.，等人（2002）Biochemistry 41，1351-1358]。负协同性与碳水化合物在其表位逐渐结合后的功能价降低有关。本研究还显示了无唾液酸胎球蛋白（ASF）（一种具有9个LacNAc表位的糖蛋白）与半乳糖凝集素-1、-2、-3、-4、-5和-7以及半乳糖凝集素-3和-5的截短单体形式（它们是动物凝集素家族的成员）的ITC结合数据的负协同性。虽然观察到ASF与半乳糖凝集素和两种截短形式结合的K-a值仅比LacNAc大50-80倍，根据观察到的宏观结合自由能和表位的降低的微观结合自由能之间的关系对数据进行分析，显示ASF的第一LacNAc表位与约6000-亲和力比最后一个表位高10倍。因此，半乳糖凝集素对ASF的第一表位的微观结合常数在纳摩尔范围内，其余表位的结合常数具有降低的梯度。结果表明，上述半乳糖凝集素与多价糖蛋白如ASF以部分高亲和力结合，与半乳糖凝集素的四级结构无关。这些发现对半乳糖凝集素与多价碳水化合物受体的结合具有重要意义。
Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K-a values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.