A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Xingxuan He,Edward H Schuchman,Calogera M Simonaro

doi:10.1590/2326-4594-jiems-2020-0021

Xingxuan He, Edward H Schuchman + Show 1 more

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https://doi.org/10.1590/2326-4594-jiems-2020-0021

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Abstract

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Highlights

Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo Syndrome type A) is a lysosomal storage disorder due to the inherited deficiency of sulfamidase (SGSH; E.C. 3.10.1.1) activity [1]
The current 4-MU SGSH assay requires two steps to measure activity: in the first step 4MU-GlcNS is desulfated by SGSH present in the test biological sample to produce 4MU-Glc, a substrate for αGD, which further converts 4MU-Glc to 4-MU in the second step
In recent years there has been considerable research around the development of disease modifying therapies for this disorder, and several clinical trials examining CNS-directed gene or enzyme replacement therapies are currently underway [4,5]. This has led to considerable research surrounding SGSH, highlighting the need for inexpensive, easy and reliable activity assays

Summary

Introduction

Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo Syndrome type A) is a lysosomal storage disorder due to the inherited deficiency of sulfamidase (SGSH; E.C. 3.10.1.1) activity [1]. SGHS is one of several enzymes included in the breakdown of the glycosaminoglycan, heparan sulfate, resulting in its accumulation in most tissues [2]. The accumulation of heparan sulfate leads to lysosomal dysfunction, chronic inflammation, cell death and disease. Due to the critical importance of heparan sulfate in brain development, MPS IIIA presents as a chronic and severe neurologic disorder [3]. Signs of the disease include speech delay, poor sleep, developmental delay, “autistic-like” behavior, and hyperactivity. It is generally diagnosed within the first decade of life, and as patients age they often experience seizures, loss of mobility, dystonia, dementia and death. No disease-specific therapies are available for the disorder, gene therapies and other approaches are under investigation [4,5]

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