Metakromatik Lökodistrofi: Üç Arilsülfataz A Mutasyonunun (P.307Glu→Lys, P.318Trp→Cys Ve C.1165G Delesyonu) Arilsülfataz A Aktivitesi ve Arilsülfataz A Proteini Üzerine Etkisinin Tanımlanması
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Metachromatic leukodystrophy (MLD) is an autosomal recessively inherited sphingolipid storage disease that occurs as a result of a lack of lysosomal enzyme Arylsulfatase A (ASA) or its activator protein. In this study, two missense mutations (c.919G?A, p.307Glu?Lys and c.954G?T, p.318Trp?Cys in exon 5) that were defined in our previous studies and a polymorphism mutation (c.1172C?G, p.391Thr?Ser in exon 7) were constructed on WT-ASA cDNA plasmid. It is shown by DNA sequence analysis that both c.919G?A and c.954G?T mutations as well as c.1165G deletion mutation were created with c.1172C?G polymorphism mutation on the WT-ASA cDNA. Plazmid DNA that was carrying mutant and normal ASA cDNA was transfered to CHO cells through transient transfection. ASA protein was produced by CHO cells. To measure the efficiency of the transfection, ASA cDNA together with hexosaminidase enzyme beta-altbirim subunit gene were cotransfected to the CHO cells. 48 hours after transfection, cells were collected, homogenized and centrifuged. After protein measurement in supernatant, ASA and hexosaminidase activity were measured. p.307Glu?Lys and p.318Trp?Cys mutations decreased ASA enzyme activity 100% compared to the control. 62 kDa sized ASA protein were detected in normal control sample, in p.307Glu?Lys mutation, and in p.318Trp?Cys mutation, and was not detected in c.1165G deletion mutation by Western blotting. This led to the thought that there is incomplet folding in mutant proteins which leads to lack of activity and that the c.1165G deletion mutation makes an important structural change that causes protein degradation. p.307Glu?Lys change in which there is an inactive protein may be a candidate for chaperone treatment. With this study, it is shown that both mutations reduce ASA activity almost completely similar to how it is in patients, and there are mutant proteins in the cell. p.307Glu?Lys and p.318Trp?Cys mutations cause late infantile form of MLD disease that has a severe clinical penotype.