Regulatory mechanism of autophagy and receptor trafficking by LAMP-2

我们已经产生了溶酶体相关膜蛋白-2(LAMP-2)缺乏的小鼠来研究该蛋白的生理作用。LAMP-2缺乏可导致约50%的早产儿死亡。所有LAMP-2缺陷小鼠中有一半。在一些LAMP-2缺乏的组织中，包括肌肉、心脏、胰腺和肝脏，观察到自噬空泡的积累。我们还观察到心肌收缩功能的降低。在这项研究中，我们扩展了培养的肝细胞的表型分析。酶活性测定表明，某些溶酶体酶向溶酶体的转运受到损害。代谢标记的组织蛋白酶D的免疫沉淀表明基因敲除细胞中的细胞内保留和加工减少。有趣的是，由于较短的半衰期，46-kDa甘露糖6-磷酸受体(MPR46)的稳态水平降低到对照组的30%。在高尔基体和内体中发现的受体较少，这表明从内体到高尔基体的循环受损。在自噬空泡中发现了更多的受体，这可能是其半衰期较短的原因。我们的数据表明，在肝细胞中，LAMP-2的缺乏直接或间接地导致MPR46的循环受损和部分溶酶体酶亚群的错误定位。由于溶酶体降解能力受损，自噬空泡可能会积聚。

We have generated mice deficient for lysosome-associated membrane protein-2 (LAMP-2) to investigate the physiological role of this protein. LAMP-2 deficiency leads to premature postnatal death of about. half of all LAMP-2 deficient mice. In several LAMP-2-deficient tissues, including muscle, heart, pancreas, and liver, an accumulation of autophagic vacuoles was observed. We also observed a reduced contractile function of the heart muscle. In this study, we extend the phenotype analysis using cultured hepatocytes. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. Interestingly, the steady-state level of 46-kDa mannose 6-phosphate receptor (MPR46) was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi and in endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of MPR46 and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulale due to impaired capacity for lysosomal degradation.