Binding of sugar ligands to Ca(2+)-dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis.
Binding of sugar ligands to Ca(2+)-dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis.
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DOI：
10.1016/s0021-9258(17)40709-5
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发表时间：
1994-06
期刊：
影响因子：
--
通讯作者：
S. Iobst;K. Drickamer
中科院分区：
文献类型：
--
作者：
S. Iobst;K. Drickamer
通过位点定向诱变，改变了大鼠血清甘露糖结合蛋白的Ca（2+）依赖性碳水化合物识别结构域（CRD），以模拟同源半乳糖结合CRD的结合位点。结合实验表明，通过3个单氨基酸变化和插入5个氨基酸片段，可以将半乳糖结合活性引入甘露糖结合位点，与asialal糖蛋白受体的CRD的结合活性几乎相同。需要单独的变化来建立与半乳糖的高亲和力结合，并通过从结合位点排除甘露糖来产生高选择性。糖- crd滴定的诱变研究和核磁共振分析表明，高亲和半乳糖结合的一个重要组成部分是糖的B面与色氨酸的相互作用。来自软骨蛋白聚糖（aggrecan）的c型CRD的结合特性也可以基于甘露糖结合CRD框架进行建模。这种较低亲和力的结合位点涉及苯丙氨酸残基对糖配体的堆积。
Changes have been introduced into the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein by site-directed mutagenesis to model the binding sites of homologous galactose-binding CRDs. Binding assays reveal that galactose-binding activity nearly identical to that of the CRD from the asialoglycoprotein receptor can be introduced into the mannose-binding site by 3 single amino acid changes and insertion of a segment of 5 amino acids. Separate changes are required to establish high-affinity binding to galactose and create high selectivity by exclusion of mannose from the binding site. The mutagenesis studies and NMR analysis of sugar-CRD titrations demonstrate that an important component of high-affinity galactose binding is interaction between the B face of the sugar and tryptophan. The binding properties of the C-type CRD from the cartilage proteoglycan, aggrecan, can also be modeled based on the mannose-binding CRD frame-work. This lower affinity binding site involves stacking of a phenylalanine residue against the sugar ligand.