A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells

摘要

A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highlydesirable for both basic research and clinical studies. NAG activity is deficient in cells from patients withMucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently availabletechniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and providea biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, celldisruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-proneprocedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedurefor measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require celldisruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that theNAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing arapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAGactivity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a masterregulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivityand reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughputscreening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation ofcandidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combinationtherapies.