Research Information System
TURKISH JOURNAL OF BIOCHEMISTRY-TURK BIYOKIMYA DERGISI, vol.34, no.3, pp.160-166, 2009 (SCI-Expanded, Scopus, TRDizin)
Objectives: We investigated the stabilizing effects of inhibiting Endoplasmic Reticulum Associated Degradation using kifunensine and lactacystin on heterologous expression of two mutant pro-forms of beta-subunit of beta-hexosaminidase, 12 bp deletion and D208N missense mutations. Methods: The Delta 12 bp and D208N mutations were introduced into beta-subunit cDNA of hexosaminidase by site-directed mutagenesis. Mutant (Delta 12 bp) or wild-type beta-subunit cDNAs were transfected to COS-1 cells. At 5(th) h of transfection, 200 mu M kifunensine or 1.5 mu M lactacystin were added to the medium. Fourty-eight hours after lysed cells were subjected to Western Blot analysis. D208N stably expressing CHO cells were grown with or without lactacystin or kifunensine in the presence of radiolabeled with S-35-Met and S-35-Cys and chased with cold media for different durations. Labeled hexosaminidase was immunoprecipitated with rabbit anti-human hexosaminidase A antibody, resolved by SDS-PAGE and visualized by autoradiography. Results: Lactacystin or kifunensine treatment of cells expressing either of the two mutants resulted in increased levels of mutant pro-beta chain. A corresponding increase in enzymatic activity was not observed in any of the mutants. Conclusion: Both mutant forms of beta-subunit are degraded through mannosidase/EDEM-1 ubiquitin/proteasome pathway. This is consistent with the model whereby beta-subunit of hexosaminidase is a substrate for the calnexin/calreticulin folding pathway in the endoplasmic reticulum. Although inhibiting either of these systems results in a increased steady-state level of mutant protein, neither leads to increased enzymatic activity.