Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A (rh-α-Gal A) is the standard treatment for Fabry disease (FD). ERT has shown a significant impact on patients; however, there is still morbidity and mortality in FD, resulting in progressive cardiac, renal, and cerebrovascular pathology. The main pathway for delivery of rh-α-Gal A to lysosome is cation-independent mannose-6-phosphate receptor (CI-M6PR) endocytosis, also known as insulin-like growth factor 2 receptor (IGF2R) endocytosis. This study aims to investigate the mechanisms of uptake of rh-α-Gal-A in different cell types, with the exploration of clathrin-dependent and caveolin assisted receptor-mediated endocytosis and the dynamics of autophagy-lysosomal functions. rh-α-Gal-A uptake was evaluated in primary fibroblasts, urine originated kidney epithelial cells, and peripheral blood mononuclear cells derived from Fabry patients and healthy controls, and in cell lines HEK293, HTP1, and HUVEC. Uptake of rh-α-Gal-A was more efficient in the cells with the lowest endogenous enzyme activity. Chloroquine and monensin significantly blocked the uptake of rh-α-Gal-A, indicating that the clathrin-mediated endocytosis is involved in recombinant enzyme delivery. Alternative caveolae-mediated endocytosis coexists with clathrin-mediated endocytosis. However, clathrin-dependent endocytosis is a dominant mechanism for enzyme uptake in all cell lines. These results show that the uptake of rh-α-Gal-A occurs rapidly and activates the autophagy-lysosomal pathway.

中文翻译：

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网格蛋白介导的重组 α-Gal-A 快速摄取至溶酶体可激活自噬。


使用重组 α-半乳糖苷酶 A (rh-α-Gal A) 的酶替代疗法 (ERT) 是法布里病 (FD) 的标准治疗方法。 ERT 对患者显示出显着的影响；然而，FD 仍然存在发病率和死亡率，导致进行性心脏、肾脏和脑血管病变。 rh-α-Gal A 递送至溶酶体的主要途径是阳离子非依赖性甘露糖 6-磷酸受体 (CI-M6PR) 内吞作用，也称为胰岛素样生长因子 2 受体 (IGF2R) 内吞作用。本研究旨在探讨不同细胞类型中 rh-α-Gal-A 的摄取机制，探索网格蛋白依赖性和小窝蛋白辅助受体介导的内吞作用以及自噬-溶酶体功能的动态。在法布里患者和健康对照的原代成纤维细胞、尿液来源的肾上皮细胞和外周血单核细胞以及细胞系 HEK293、HTP1 和 HUVEC 中评估了 rh-α-Gal-A 的摄取。在内源酶活性最低的细胞中，rh-α-Gal-A 的摄取效率更高。氯喹和莫能菌素显着阻断 rh-α-Gal-A 的摄取，表明网格蛋白介导的内吞作用参与重组酶递送。替代性小窝介导的内吞作用与网格蛋白介导的内吞作用共存。然而，网格蛋白依赖性内吞作用是所有细胞系中酶摄取的主要机制。这些结果表明 rh-α-Gal-A 的摄取发生迅速并激活自噬-溶酶体途径。