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dc.contributor.authorMauri, Victor
Lotfi, Parisa
Segatori, Laura
Sardiello, Marco
dc.date.accessioned 2013-07-12T19:49:05Z
dc.date.available 2013-07-12T19:49:05Z
dc.date.issued 2013
dc.identifier.citation Mauri, Victor, Lotfi, Parisa, Segatori, Laura, et al.. "A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells." PLoS One, 8, no. 6 (2013) Public Library of Science: e68060. http://dx.doi.org/10.1371/journal.pone.0068060.
dc.identifier.urihttps://hdl.handle.net/1911/71546
dc.description.abstract A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for 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 cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies.
dc.language.iso eng
dc.publisher Public Library of Science
dc.rights This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.title A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells
dc.type Journal article
dc.contributor.funder Virginia and L.S. Simmons Family Foundation
dc.contributor.funder Welch Foundation
dc.citation.journalTitle PLoS One
dc.citation.volumeNumber 8
dc.citation.issueNumber 6
dc.embargo.terms none
dc.type.dcmi Text
dc.identifier.doihttp://dx.doi.org/10.1371/journal.pone.0068060
dc.identifier.pmcid PMC3695942
dc.identifier.pmid 23840811
dc.identifier.grantID Collaborative Research Fund (Virginia and L.S. Simmons Family Foundation)
dc.type.publication publisher version
dc.citation.firstpage e68060


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