ANKYRIN BINDS TO THE 15TH REPETITIVE UNIT OF ERYTHROID AND NONERYTHROID BETA-SPECTRIN
ANKYRIN BINDS TO THE 15TH REPETITIVE UNIT OF ERYTHROID AND NONERYTHROID BETA-SPECTRIN
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DOI：
10.1083/jcb.115.1.267
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发表时间：
1991-10-01
影响因子：
7.8
通讯作者：
MORROW, JS
中科院分区：
文献类型：
--
作者：
KENNEDY, SP;WARREN, SL;MORROW, JS
在红系和非红系细胞中，锚蛋白通过与血影蛋白的β亚基结合来介导血影蛋白与跨膜整合蛋白的结合。以前使用酶消化、2-硝基-5-硫氰基苯甲酸裂解和旋转阴影技术的研究已经将血影蛋白-锚蛋白结合位点放置在β-血影蛋白的COOH-末端三分之一，但确切的位置尚不清楚。我们利用谷胱甘肽S转移酶原核表达系统，从编码红系和非红系β-血影蛋白羧基末端大约一半的基因中制备了重组的红系和非红系β-血影蛋白。重组血影蛋白与I-125标记的天然血影蛋白以等摩尔比的方式与红细胞膜囊泡(IOV)结合，并通过沉降速度实验测定其在体外与锚定蛋白的结合。尽管全长的β-血影蛋白可以抑制所有的血影蛋白与IOV的结合，但含有完整的锚蛋白结合区但缺少分子氨基末端一半的重组β-血影蛋白却不能抑制大约25%的IOV的结合能力，这表明不依赖于锚蛋白的血影蛋白膜结合部位必须位于β-血影蛋白的氨基末端。通过核酸酶消化来自锚蛋白结合结构物的β-血影蛋白cDNA，产生一组缩短的重组子嵌套。这些都定义了锚蛋白结合域，包括红系和非红系β-血影蛋白的第15个重复单位，红系β-血影蛋白的1,768-1,898个氨基酸残基。锚蛋白结合重复单元是非典型的，因为它在重复序列中的45(1,811)位缺少保守的色氨酸，并且在重复序列的第三末端含有一个非同源的43个残基片段。该重复序列的前30个残基在红系和非红系β-光谱蛋白之间高度保守，对锚蛋白结合活性至关重要。我们假设Ankyrin直接与红系和非红系β-血影蛋白第15个重复单元中的非同源片段结合，但该序列必须在适当折叠的血影蛋白“重复单元”结构的背景下呈现。未来的研究将确定重复单元中的哪些残基是活性所必需的，以及哪些残基决定了不同形式的锚定蛋白的不同光谱蛋白的特异性。
Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with I-125-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25 % of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.