Investigation On The Molecular Mechanism Of GLA Mutation Causing Fabry Disease

In the previous study,our group has identified four GLA mutations from four Fabry disease patients through Sanger sequencing,namely c.119C>A(p.P40H),c.280T>C(C94R),c.680G>C(p.R227P)and c.801+1G>A(p.L268 fsX3).To further reveal the molecular mechanism of GLA gene mutation causing Fabry disease,the effects of GLA gene mutations on protein structure were analyzed through bioinformatics methods and found that the altered amino acids are all close to the enzyme activity center,which may alter the enzyme activity leading to the disease.The mutant plasmids containing the above four mutations were constructed by site-directed mutagenesis and immunofluorescence staining were performed to observe the localization of GLA mutants.Native gel electrophoresis was performed to analyse the formation of GLA homodimer and the results showed that GLA c.280T>C(C94R)and c.801+1G>A(p.L268 fsX3)mutations lead to the accumulation of GLA mutants in the cytoplasm without affecting the formation of GLA homodimers.The GLA knockdown and stable overexpression of GLA wildtype and mutants HEK293T cell lines were establised by lentivirus packaging system and the enzyme activity ofα-galactosidase a activitited were determined.The results showed that all the GLA mutants led to the reduction of α-galactosidase A activity.Subsequently,the effect of GLA mutation on autophagy was detected in GLA mutants overexpressing cells after starvation.The results showed that the numbers of autophagosomes were significantly increased 3 hours and 6 hours post-starvation;when the starvation treatment extended upto 9 hours,the amount of LC3-Ⅱ in GLA wild-type overexpressing cells was decreased to the normal level.However,in GLA mutants overexpressing cells,the amounts of LC3-Ⅱ were still remained at a high level after 9 hours’ starvation treatment,indicating that the overexpression of GLA mutants caused autophagosomes accumulation in the cell which inturn upregulated the phosphorylation of mTOR to reduce the formation of autophagosomes.GLA mutation results in the increase of mTOR phosphorylation,on the one hand,inhibiting the formation and reducing the accumulation of autophagosomes,on the other hand,contributing to the reformation of autolysosomes to lysosomes and promoting the expression of lysosomal-related proteins LAMP1 and LAMP2,resulting in an increase in the number and activity of lysosomes.Co-immunoprecipitation combined with mass spectrometry showed that GLA interacted with autophagy related protein ATG4 C,indicating an important roles in the regulation of the autophagy damage caused by GLA gene mutation.In conclusion,the study focused on the GLA gene mutation from the patients and revealed the molecular mechanism of GLA mutation leading to Fabry’s disease through a series of experiments.Our data will further provide theoretical basis for the molecular diagnosis and drug development of therapeutic targets on Fabry disease.