Leg1蛋白分子修饰解析及其在肝脏发育和饥饿生理反应中的作用机理研究

The liver expresses a large number of liver-specific or -enriched genes which encode factors essential for the liver function. Loss-of-function of such genes often causes diseases in human. Although vast information has been obtained for these liver genes their roles in the early liver development are less studied. Based on analyzing the microarray data of liver-enriched genes in zebrafish we were the first to identify a novel gene leg1 (liver-enriched gene 1). Our further studies revealed that leg1 has two gene copies, leg1a and leg1b, which are closely linked to each other on chromosome 20. Both leg1a and leg1b were expressed in the adult liver. The leg1 gene encodes a novel secretory protein. Recently, our unpublished data showed that 1) loss of maternal and zygotic Leg1 protein resulted in a growth environment-dependent small liver phenotype in zebrafish; 2) we also found that Leg1 protein is modified by glycosylation and disulfide bond; 3) more interestingly, we found that the expression of leg1 can be iduced by starvation treatment in both zebrafish and mouse. Database search and sequence alignment showed that Leg1 evolutionally appears to be unique in the vertebrates, however, the function of Leg1 has not been well studied. Here we designed a detailed proposal to study the biological function of Leg1 including 1) the molecular mechanism of Leg1 in regulating liver development, 2) the relationship between Leg1 protein modification and liver development, and 3) the role of Leg1 during starvation response in zebrafish and mouse. We believe that the result obtained will no doubt extend our understanding of the process of liver development and the physiology of starvation response.

成体肝脏表达多种肝脏特异或富集的基因，这些基因的缺陷往往会造成人类疾病。尽管目前人们对这类基因的生理功能已有了较多的了解，但对它们是否参与调控肝脏发育知之甚少。通过分析斑马鱼肝脏富集基因的基因芯片数据我们首次鉴定了新基因leg1。斑马鱼的leg1基因有两个拷贝leg1a和leg1b，它们在染色体上紧密连锁，并均在成体肝脏中富集表达。leg1编码一个全新的外分泌蛋白。我们最新的未发表的数据显示leg1a母源-合子突变体呈现生长环境依赖性小肝脏性状、Leg1蛋白分子存在糖基化和二硫键修饰，且leg1转录表达在斑马鱼和小鼠中均受饥饿诱导。本研究计划深入研究Leg1调控肝脏发育的机制、leg1蛋白分子修饰与其功能关系、Leg1在饥饿生理反应中的作用。研究成果不仅将极大地丰富肝脏早期发育的内涵，同时还有可能为饥饿生理学开辟新的研究方向。

本项目四年研究计划主要是围绕解析斑马鱼Leg1蛋白的生物学功能开展深入研究，同时初步探索小鼠mLeg1表达的组织专一性及其对饥饿的响应。本项目研究目标是精确定位Leg1糖苷化及二硫键修饰的氨基酸残基，揭示Leg1 修饰对Leg1 分泌及对肝脏发育得影响，由此阐明Leg1 在肝脏发育中的功能及其分子机理；同时，我们还利用斑马鱼和小鼠两种不同动物模式中揭示Leg1 在饥饿应答中的作用，探索在进化过程中肝脏和唾液腺在饥饿生理反应上的相关性。在项目执行过程中，绝大部分年度研究计划已顺利完成，且很多工作已超额完成，这些成果为下一阶段的工作打下了坚实的基础。.主要成果总结如下：.1）leg1基因仅在脊椎动物中被发现，该基因编码一个相对保守的全新的外分泌蛋白。斑马鱼的leg1基因有两个拷贝（leg1a和leg1b），两个同源基因在染色体上紧密连锁，且均在肝脏中富集表达。在本项目执行期间，我们发现斑马鱼Leg1作为一个新的内分泌信号因子介导下游的抗氧化途径，该信号途径在低温季节保护斑马鱼的生长发育。我们还发现斑马鱼Leg1受到糖基化修饰，且该糖基化修饰为其功能所必需。该项工作于2016年2月22日发表在PLoS Genetics (12(2): e1005881)(十分抱歉，忘了标注），该研究结果及进一步的深入研究有望揭示硬骨鱼类适应环境改变的原因。.2）此外，我们发现在小鼠中mLeg1并非由肝脏表达，而是由唾液腺细胞表达，其在唾液腺的表达受饥饿后再进食的诱导，且mLeg1分泌到唾液中,因此是一个外分泌因子。有趣的是mLeg1具抗胃酸能力，且似乎能通过肠壁进入血液循环，然后通过EGFR-PI3K-Akt-Srebp1c信号通路促进肝脏细胞的脂肪酸合成反应。我们还发现mLeg1参与调控高脂食物诱导的肥胖症。这方面的研究成果获得四项授权发明专利。