Characterization of the βγ-crystallin domains of βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex, from the skin of the toad Bombina maxima
Characterization of the βγ-crystallin domains of βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex, from the skin of the toad Bombina maxima
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DOI：
10.1016/j.biochi.2011.07.013
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发表时间：
2011-10-01
期刊：
影响因子：
3.9
通讯作者：
Zhang, Yun
中科院分区：
文献类型：
--
作者：
Gao, Qian;Xiang, Yang;Zhang, Yun
β - γ - cat是一种天然存在的72 kda复合物，由非晶态β - γ -晶体蛋白（α -亚基，cat - α）和三叶因子（β -亚基，cat - β）组成，含有α - β的非共价连接形式(2)，从蟾蜍bomina maxima的皮肤分泌物中分离出来。cat - α的N端区域（cat - α N，残基1-170）含有两个β - γ -晶体蛋白结构域，而C端区域（cat - α C）与产气荚膜梭菌毒素的膜插入结构域序列同源。为了研究β - γ - cat的β - γ -结晶蛋白结构域的生化特性，我们在大肠杆菌中表达了cat - α N、cat - α C和cat - β。自然组装的β - γ - cat的共免疫沉淀证实了cat - α和cat - β复合物始终存在。此外，重组cat - β结合的重组cat - α N在cat - α N中发现了Ca（2+）结合基元，重组cat - α N能够结合钙探针铽。然而，Ca（2+）结合后，cat - α N的构象没有明显改变。β - γ - cat对人红细胞具有很强的溶血活性，用β - γ - cat处理红细胞可导致Ca（2+）快速内流，最终导致溶血。然而，在缺乏细胞外Ca（2+）的情况下，尽管在红细胞膜中观察到β - γ - cat的结合和低聚化，但未检测到明显的溶血。我们的数据证明了cat - β（三叶因子）与cat - α N （β - γ -晶体蛋白结构域）的结合，并为体内β - γ -晶体蛋白和三叶因子复合物的形成提供了基础。此外，β - γ - cat的β - γ -晶体蛋白结构域能够结合Ca(2+)， β - γ - cat诱导的溶血依赖于Ca（2+）。(C) 2011 Elsevier Masson SAS。版权所有。
beta gamma-CAT is a naturally existing 72-kDa complex of a non-lens beta gamma-crystallin (alpha-subunit, CAT-alpha) and a trefoil factor (beta-subunit, CAT-beta) that contains a non-covalently linked form of alpha beta(2) and was isolated from the skin secretions of the toad Bombina maxima. The N-terminal region of CAT-alpha (CAT-alpha N, residues 1-170) contains two beta gamma-crystallin domains while the C-terminal region (CAT-alpha C) has sequence homology to the membrane insertion domain of the Clostridium perfringens epsilon toxin. To examine the biochemical characteristics of the beta gamma-crystallin domains of beta gamma-CAT, CAT-alpha N, CAT-alpha C and CAT-beta were expressed in Escherichia coli. Co-immunoprecipitation of the naturally assembled beta gamma-CAT confirmed that the CAT-alpha and CAT-beta complex always exists. Furthermore, recombinant CAT-beta bound recombinant CAT-alpha N. Ca(2+)-binding motifs were identified in CAT-alpha N, and recombinant CAT-alpha N was able to bind the calcium probe terbium. However, the conformation of CAT-alpha N was not significantly altered upon Ca(2+) binding. beta gamma-CAT possesses strong hemolytic activity toward human erythrocytes, and treatment of erythrocytes with beta gamma-CAT resulted in a rapid Ca(2+) influx, eventually leading to hemolysis. However, in the absence of extracellular Ca(2+), no significant hemolysis was detected, even though the binding and oligomerization of beta gamma-CAT in the erythrocyte membrane was observed. Our data demonstrate the binding of CAT-beta (a trefoil factor) to CAT-alpha N (beta gamma-crystallin domains) and provide a basis for the formation of a beta gamma-crystallin and trefoil factor complex in vivo. Furthermore, the beta gamma-crystallin domains of beta gamma-CAT are able to bind Ca(2+), and beta gamma-CAT-induced hemolysis is Ca(2+) dependent. (C) 2011 Elsevier Masson SAS. All rights reserved.