Analysis of heparin, α-dystroglycan and sulfatide binding to the G domain of the laminin α1 chain by site-directed mutagenesis
Analysis of heparin, α-dystroglycan and sulfatide binding to the G domain of the laminin α1 chain by site-directed mutagenesis
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DOI：
10.1006/jmbi.1999.2606
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发表时间：
1999-03-26
影响因子：
5.6
通讯作者：
Timpl, R
中科院分区：
文献类型：
--
作者：
Andac, Z;Sasaki, T;Timpl, R
395个残基的蛋白水解性片段E3，包括小鼠层粘连蛋白α1链的两个最大的C-末端LG模块，先前被证明包含与肝素、α-营养不良聚糖和硫脂的主要结合部位。相同的片段(α1LG4-5)及其单独的α1LG4和α1LG5模块现已在哺乳动物细胞中通过重组生产获得。圆二色谱、电子显微镜和免疫学分析表明，这些片段明显折叠成天然形式。片段Alpha 1LG4-5对肝素、α-抗肌营养不良聚糖和硫脂的结合力比E3好五到十倍。这些结合活性可以专门定位于阿尔法1LG4模块。侧链修饰和蛋白质降解表明，α1LG4 C末端区域的Lys和Arg残基是肝素结合所必需的。14个单到三个点突变证实了这一点，它们确定了三个不连续的碱性区域(2766-2770,2791-2793,2819-2820)与肝素和硫脂结合有关。这些区域中的两个也被先前被证明抑制肝素结合的单抗识别。同样的三个区域和几个额外的碱性残基也对细胞受体α-肌营养不良聚糖的结合做出了主要贡献，表明有更大的结合表位。这些数据也与之前的发现一致，即肝素竞争α-肌营养不良聚糖结合。(C)1999年学术出版社。
The 395-residue proteolytic fragment E3, which comprises the two most C-terminal LG modules of the mouse laminin alpha 1 chain, was previously shown to contain major binding sites for heparin, alpha-dystroglycan and sulfatides. The same fragment (alpha 1LG4-5) and its individual alpha 1LG4 and alpha 1LG5 modules have now been obtained by recombinant production in mammalian cells. These fragments were apparently folded into a native form, as shown by circular dichroism, electron microscopy and immunological assays. Fragment alpha 1LG4-5 bound about five- to tenfold better to heparin, alpha-dystroglycan and sulfatides than E3. These binding activities could be exclusively localized to the alpha 1LG4 module. Side-chain modifications and proteolysis demonstrated that Lys and Arg residues in the C-terminal region of alpha 1LG4 are essential for heparin binding. This was confirmed by 14 single to triple point mutations, which identified three non-contiguous basic regions (positions 2766-2770, 2791-2793, 2819-2820) as contributing to both heparin and sulfatide binding. Two of these regions were also recognized by monoclonal antibodies which have previously been shown to inhibit heparin binding. The same three regions and a few additional basic residues also make major contributions to the binding of the cellular receptor alpha-dystroglycan, indicating a larger binding epitope. The data are also consistent with previous findings that heparin competes for alpha-dystroglycan binding. (C) 1999 Academic Press.