Degradation Of ALG11-CDG Mutant Protein Is Regulated By The Proteasome Pathway(ERAD)

N-glycosylation is one of the most important post-translational modification processes in eukaryotic cells that directly affects the structural and biological functions of glycoproteins.The synthesis of N-glycan starts from the endoplasmic reticulum(ER),where a highly conserved dolichol linked oligosaccharide precursor(Glc3-Man9-Glc NAc2-PP-Dol)was sequensualy assembled by a series of ALG(Asparagine-linked Glycosylation)family glycosyltransferases.Mutations in ALG family genes will cause related congenital glycosylation disease,namely ALG-CDG(ALG-related Congenital Disorders of Glycosylation).Alg11 glycosyltransferase catalyzes the transfer of the 4th and 5th ?-1,2 mannose(Man)to the substrate Man3-Gn2-PP-Dol to produce Man5-Gn2-PP-Dol.ALG11-CDG causes broad multisystem defects,nearly all have neurological involvement.Current study on ALG11-CDG is more focused on discovering its clinical symptoms.No much atteintion is paid on the properties and functions of the mutant Alg11 proteins.Using the cell-molecular and biochemical approaches,we have challenged to explore the degradation pathway of Alg11 and its CDG mutant proteins.Our results can be list as follow:(1)Comparing expression levels of ALG11-CDG mutant protein in embryonic kidney epithelial cells(HEK293),Saccharomyces cerevisiae and E.coli,we found that ALG11-CDG mutant protein is unstable and may be recognized and degraded by the protein degradation pathway in eukaryotic cells.(2)Using proteasome inhibitor and lysosomal inhibitor,we found that the degradation of the Alg11 wild type and ALG11-CDG mutant protein is related to the proteasome pathway.(3)By knocked out ubiquitin ligases in HEK293,we found that the degradation of Alg11 wild-type and ALG11-CDG mutant proteins is related to the ERAD-C pathway mediated by the E3 ligase MARCH6,and its degradation determinant may be located on the cytoplasmic side of the endoplasmic reticulum.Our results are not only important for uncovering the molecular mechanism of Alg11 protein degradation system in eukaryotic cells,but also providing new ideas for the diagnosis and therapeutics of ALG11-CDG.