Enzyme Unit Great Ormond Street Hospital, London WC1N 3JH, UK.
University College London Great Ormond Street Institute of Child Health London, London WC1N 1EH, UK.
Int J Mol Sci. 2019 Sep 5;20(18):4349. doi: 10.3390/ijms20184349.
Fabry disease (FD) is caused by mutations in the gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.
法布里病（FD）是由编码溶酶体α-半乳糖苷酶-A（α-gal-A）的基因发生突变引起的。已经提出了许多致病机制，包括线粒体呼吸链活性丧失。对于 FD，基因治疗开始被用作一种治疗方法。鉴于 FD 中报道的线粒体功能丧失，我们在这里考虑了线粒体呼吸链活性丧失对慢病毒载体增加细胞 α-gal-A 活性和参与交叉校正的能力的影响。本研究使用 Jurkat 细胞，并暴露于不断增加的病毒拷贝数。然后在存在或不存在线粒体复合物 I 抑制剂鱼藤酮的情况下测定细胞内和细胞外酶活性。还评估了细胞摄取释放酶的能力。随着转基因拷贝数的增加，细胞内α-gal-A 活性增加，但这与 Km 值增加有关。释放的酶和细胞摄取也得到了证实。然而，在鱼藤酮存在的情况下，酶的释放被抑制了 37%。过量的酶生成可能导致具有较差动力学特性的蛋白质，而背景下受损的线粒体功能可能会损害交叉校正过程。
