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.
Highlights
Mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo syndrome B, OMIM #252920) is an autosomal recessive lysosomal storage disorder (LSD) caused by mutations in the gene encoding the lysosomal hydrolase, N-alpha-acetylglucosaminidase (NAGLU or NAG; E.C. 3.2.1.50)
Fibroblasts are in a metabolic state in which highly differentiated cellular functions, including the synthesis of lysosomal hydrolases, become more important than cell division [55]
After a 17-hour incubation period, the reaction was stopped with a glycine buffer at pH 10.8 and the released fluorescence was measured on a plate reader using an excitation wavelength of 360 nm and measuring the emission at 460 nm [30]
Summary
Mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo syndrome B, OMIM #252920) is an autosomal recessive lysosomal storage disorder (LSD) caused by mutations in the gene encoding the lysosomal hydrolase, N-alpha-acetylglucosaminidase (NAGLU or NAG; E.C. 3.2.1.50). Substrate reduction therapy (SRT) aims at reducing the synthesis of the specific substrate that accumulates in the patient’s cells due to the catabolic enzyme deficiency [12]. Because it is based on the use of small molecules that can potentially cross the BBB, SRT represents a promising strategy to address CNS symptoms in neuropathic forms of LSDs [13]. Gene therapy (GT) is an attractive option for MPS because it exploits the principle of crosscorrections–enzymes produced by the transduced cells are secreted and taken up by surrounding cells, including non-transduced cells, via the M6PR pathway, correcting cellular storage [17,18,19]. TFEB can enhance the clearance of pathogenic storage material and counteract disease progression, a principle that is being demonstrated in multiple models of neurodegenerative diseases including LSDs, Huntington disease, Alzheimer disease and Parkinson disease [20,23,24,25,26,27]
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