大鼠Map1LC3家族两个新基因的克隆及功能初步研究

【作者】 吴家雪；

【导师】 胡新文； 余龙；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2003， 硕士

【摘要】 大鼠微管相关蛋白轻链3(Map1lc3)蛋白在体内同时以两种不同分子量的形式存在。其中18KDa的Map1lc3蛋白是微管相关蛋白1的轻链亚基，称为Lc3-Ⅰ；另一种16KDa的Map1lc3蛋白则是自噬体膜的必须组分，称为Lc3-Ⅱ。Lc3-Ⅰ和Lc3-Ⅱ都是一系列翻译后修饰的产物，其中特征性的修饰是在保守Gly120后发生的羧基端切割，此切割修饰对Lc3-Ⅱ定位于自噬体膜尤为重要。我们通过生物信息学方法克隆到大鼠LC3的两个新的同源基因，分别命名为大鼠Map1LC3A和Map1LC3B，其中Map1LC3B是原Map1lC3的一个可变剪接本。分析大鼠Map1lC3家族两个新成员的组织表达谱，结果显示它们的组织表达谱有所差异，提示这两个新基因可能在不同的组织发挥功能。二个同源基因的推导蛋白与大鼠Map1lc3蛋白高度保守。分别在人的HEK293细胞中表达大鼠Map1lc3A和Map1lc3B，western blotting结果显示两者在体内皆以18KD和16KD两种大小不同的蛋白形式存在(LC3A-Ⅰ和LC3A-Ⅱ；LC3B-Ⅰ和LC3B-Ⅱ)，而且18KD的LC3A/B—Ⅰ蛋白的表达量明显多于相应的16KD的LC3A/B—Ⅱ蛋白。进一步的突变分析表明，Gly(120)是两个蛋白在体内发生翻译后修饰的关键位点。另外，通过荧光定位以及与MDC的共定位分析发现两个基因与大鼠的LC3相似，也是定位在自噬体上。这两个基因的克隆将有助于揭示自噬体在生物体内的形成过程及功能。更多还原

【Abstract】 Two forms of rat Microtubule-Associated Proteinl Light Chain 3 (Mapllc3) exist and serve as a subunit of the microtubule-associated protein 1 and an essential constituent of autophagosome membranes, respectively. In vivo, the two functional forms of rat Mapllc3 with apparent mobilities of 18 and 16KDa, termed Lc3-I and Lc3-II, separately, were produced by a series of post-translational modifications including a characteristic C-terminal cleavage after the conserved Glyl20 residue and this cleavage is essential for the membrane association of the 16KDa rat Map1lc3 protein., we cloned two novel isoforms of rat Map1LC3 by bioinformatics and named MapILCSA and Map1LC3B, respectively. The tissue distribution of transcription of two genes is different. In addition, They were transfected into human HEK293T cells and analyzed by immunoblotting, suggesting that two forms of posttranslational modification existed in rat MaplLC3A/B, which is similar to that found in rat Mapllc3 and yeast Apg8p. Mutational and deletion analysis indicated that the conserved Gly residue is crucial for the post-translational modifications of MaplLC3A and B. Moreover, the amounts of larger MAP1LC3A and B proteins were more abundant than that of smaller MAP1C3A and B proteins, respectively. Subcellular localization by immunofluorescence revealed that two rat isoforms are associated with the autophagic pathway. These results are help to discover the mechanisms and functions of autophagy in vivo.更多还原