Exploring the microtubule-binding region of bovine microtubule-associated protein-2 (MAP-2): cDNA sequencing, bacterial expression, and site-directed mutagenesis.
Exploring the microtubule-binding region of bovine microtubule-associated protein-2 (MAP-2): cDNA sequencing, bacterial expression, and site-directed mutagenesis.
复制标题
探索牛微管相关蛋白 2 (MAP-2) 的微管结合区域：cDNA 测序、细菌表达和定点诱变。
DOI：
10.1021/bi00249a006
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Purich,DL
中科院分区：
文献类型：
--
作者：
Coffey,RL;Joly,JC;Cain,BD;Purich,DL
1994年7月26日接收的修订版Mandarin pt ®摘要：对编码牛微管相关蛋白-2（MAP-2）微管结合区（MTBR）的牛MAP-2 cDNA的1.1内切酶片段进行测序。相对于小鼠、大鼠和人MAP-2，我们观察到显著的一级结构保留，甚至超过了负责微管结合相互作用的三个不同肽重复序列的序列和间隔。为了进一步利用定点突变分析微管-MAP相互作用，我们开发了一个细菌表达系统，编码MAP-2的MT结合片段，起始于凝血酶切割位点（位置1629），并继续到C-末端。该MT结合片段通过利用该细胞基质组分的不寻常的热稳定性和等电特性纯化至均一。我们发现，MT结合域容易促进微管蛋白聚合，并且在该重组蛋白的存在下，临界微管蛋白浓度降低。由于第二个重复序列类似物可以促进微管蛋白聚合，以及取代MT结合区的MAP-2，这项研究的目的是了解更多的重要性，每个重复序列在MT结合。因此，我们突变第一和第三序列以类似于第二重复序列，从而产生命名为mi2-m2-m33、mi-m32和mi2-m2-m32的突变体。这些重组蛋白的结合亲和力与野生型MT结合片段的相同浓度相当或稍好。同样，当第一或第三序列被第二个十八肽重复序列的精确拷贝取代时，结合亲和力几乎没有增加，如果有的话，这反映在突变MT结合片段促进微管蛋白聚合的能力上。因此，我们可以得出结论，与三个第二序列重复中的每一个相关的结合能不是相加的，并且这些发现加强了第二序列重复在微管结合中起主导作用的结论。微管相关蛋白-2，神经元树突过程的细胞基质组分，与微管（MT）1相互作用，并被认为通过在微管之间形成交联来稳定神经元细胞骨架（Olmsted，1986）。一个单一的MAP-2基因似乎作为一个共同的MAP-2 mRNA前体的选择性剪接产生的几种不同的蛋白质种类的前体。MAP-2ab是出现在成年神经元树突中的高分子量形式（Papalikopoulou等人，1989），MAP-2c是一种低分子量对应物，含有与成虫形式相同的极端氨基和羧基末端。MAP-2ab可以容易地被丝氨酸蛋白酶（Vallee和Borisy，1977）切割成N-末端突出臂结构域和C-末端微管结合区，后者在此称为MAP-2 MTBR。然而，与胰凝乳蛋白酶和胰蛋白酶不同的是，丝氨酸特异性蛋白酶凝血酶在切割胰凝乳蛋白酶后产生稳定的极限消化产物。
Revised Manuscript Received July 26, 1994® abstract: A 1.1 kilobase fragment of bovine microtubule-associated protein-2 (MAP-2) cDNA coding for bovine MAP-2 microtubule-binding region (MTBR) was sequenced. Relative to mouse, rat, and human MAP-2, we observed striking preservation of primary structure, even beyond the sequence and spacing of the three nonidentical peptide repeats responsible for microtubule-bindinginteractions. For further analysis of microtubule—MAPinteractions using site-directed mutagenesis, we developed a bacterial expression system coding for the MT-binding fragment of MAP-2 starting at the thrombin cleavage site (position 1629) and continuing tothe C-terminus. This MT-binding fragment was purified to homogeneity by taking advantage of the unusual heat-stability and isoelectric properties of this cytomatrix component. We found that the MT-binding domain readily promoted tubulin polymerization, and the critical tubulin concentration was reduced in the presence of this recombinant protein. Because a second repeated sequence analogue can promote tubulin polymerization as well as displace the MT-binding region of MAP-2, this study was designed to learn more about the importance of each repeated sequence in MT binding. Accordingly, we mutated the first and third sequences to resemble the second repeated sequence, thereby generating the mutants designated mi2-m2-ni3, mi-ni32, andmi2-m2-m32. Theserecombinant proteins bound with an affinity comparable to or slightly betterthan equal concentrations of wild-type MT-binding fragment. Likewise, when the first or third sequence was replaced by an exact copy of the second octadecapeptide repeat, there was little, if any, increase in binding affinity, as reflected in the ability of mutant MT-binding fragmentsto promote tubulin polymerization. We can thus conclude that the binding energies associated with each of the three second-sequence repeats were not additive, and these findings fortify the conclusion that the second sequence repeat plays a dominant role in microtubule binding.Microtubule-associated protein-2, a cytomatrix component of neuronal dendritic processes, interacts with microtubules (MTs) 1 and is thought to stabilize the neuronal cytoskeleton by forming cross-links between microtubules (Olmsted, 1986). A single MAP-2 gene appears to serve as a precursor for several different protein species that arise from alternative splicing of a common MAP-2 mRNA precursor. MAP-2ab are high-molecular-weight forms appearing in dendrites of adult neurons(Papandrikopoulou et al., 1989), and MAP-2c is a low-molecular-weight counterpart containing the same extreme amino and carboxytermini found in the adult forms. MAP-2ab can be readily cleaved by serine proteases (Vallee & Borisy, 1977) into on N-terminal projection-arm domain and a C-terminal microtubule-binding region, the latter designated here as MAP-2 MTBR. Unlike chymotrypsin and trypsin, however, the arginine-specific protease thrombin produces stable limit digestion products after cleavageat the