hymecromone and Sandhoff-Disease

Tay-Sachs and Sandhoff diseases are lysosomal storage disorders that result from an inherited deficiency of beta-hexosaminidase A (alphabeta). Whereas the acute forms are associated with a total absence of hexosaminidase A and early death, the chronic adult forms exist with activity and protein levels of approximately 5%, and unaffected individuals have been found with only 10% of normal levels. Surprisingly, almost all disease-associated missense mutations do not affect the active site of the enzyme but, rather, inhibit its ability to obtain and/or retain its native fold in the endoplasmic reticulum, resulting in its retention and accelerated degradation. By growing adult Tay-Sachs fibroblasts in culture medium containing known inhibitors of hexosaminidase we have raised the residual protein and activity levels of intralysosomal hexosaminidase A well above the critical 10% of normal levels. A similar effect was observed in fibroblasts from an adult Sandhoff patient. We propose that these hexosaminidase inhibitors function as pharmacological chaperones, enhancing the stability of the native conformation of the enzyme, increasing the amount of hexosaminidase A capable of exiting the endoplasmic reticulum for transport to the lysosome. Therefore, pharmacological chaperones could provide a novel approach to the treatment of adult Tay-Sachs and possibly Sandhoff diseases.

Topics: Adult; beta-N-Acetylhexosaminidases; Cell Line; Enzyme Activation; Enzyme Inhibitors; Female; Fibroblasts; Hexosaminidase A; Hot Temperature; Humans; Hymecromone; In Vitro Techniques; Lysosomes; Molecular Chaperones; Mutation; Protein Folding; Sandhoff Disease; Tay-Sachs Disease

We have adapted two methods to evaluate the beta-hexosaminidase (HEX) enzymatic activity in cultured cells, based on the use of (i) the fluorogenic substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2- deoxy-beta-D-glucopyranoside (MU-GlcNAc-6-SO4) and (ii) the naphthol AS-BI-N-acetyl-beta-D-glucosaminide and hexazotized pararosaniline. We demonstrate that both methods could be used for the HEX isoenzymes by comparing wild-type and mutant human fibroblast cell lines, deficient for either an alpha or beta subunit from Tay-Sachs and Sandhoff patients. This in situ cytochemical assessment of HEX activity offers a rapid evaluation to study the expression of this enzyme in a heterogeneous cell population such as in gene transfer experiments.

Topics: Acetylglucosamine; beta-N-Acetylhexosaminidases; Cells, Cultured; Fibroblasts; Glucuronates; Histocytochemistry; Humans; Hymecromone; Isoenzymes; Mutation; Naphthols; Sandhoff Disease; Spectrometry, Fluorescence; Tay-Sachs Disease

1. In human plasma, an enzyme is present which hydrolyzes 4-methylumbelliferyl-tetra-N-acetylchitotetraoside. The function of this enzyme is unknown. 2. We have examined whether hyaluronidase, neutral endoglucosaminidase, N-acetyl-beta-D-hexosaminidase, aspartylglucosaminidase, beta-D-glucosidase, and chitobiase could hydrolyze MU-TACT. The results obtained are detailed below. 3. A purified commercial preparation of hyaluronidase does not hydrolyze MU-TACT. 4. Substrate specificity requirements, pH optimum and subcellular localization indicate that neutral endoglucosaminidase is distinguishable from MU-TACT hydrolase. Also commercial neutral endoglucosaminidase D and H have no affinity towards MU-TACT. 5. N-Acetyl-beta-D-hexosaminidase is different from MU-TACT hydrolase for the following reasons: (a) a purified enzyme preparation does not hydrolyze MU-TACT; (b) there is no correlation in the activity of the enzymes; (c) MU-TACT hydrolase is not deficient in cells of a patient with a deficiency of total N-acetyl-beta-D-glucosaminidase; and (d) the 2 enzymes have very different chromatographic characteristics and Con A binding properties. 6. Enzyme characteristics, substrate structural requirements and a lack of correlation with MU-TACT hydrolase activity suggest that aspartylglucosaminidase, beta-D-glucosidase, and chitobiase are not involved in the hydrolysis of MU-TACT. 7. None of the enzymes which we have considered corresponds to MU-TACT hydrolase. The exact nature and the function of the enzyme remains an enigma.

Topics: Animals; Carbohydrate Sequence; Cell Fractionation; Cells, Cultured; Female; Glucosidases; Humans; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Liver; Molecular Sequence Data; Oligosaccharides; Rats; Rats, Wistar; Sandhoff Disease

Topics: Amniotic Fluid; Female; Hexosaminidases; Humans; Hymecromone; Pregnancy; Prenatal Diagnosis; Sandhoff Disease; Tay-Sachs Disease; Umbelliferones

4-Methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized and tested as a substrate for the diagnosis of GM2 gangliosidoses using leukocytes. Less than 2% of normal activity was measured in homogenates from patients with typical Tay-Sachs disease and from a patient with a variant form having 37% Hexosaminidase A by heat denaturation using the usual fluorogenic substrate. An adult patient had 8.5% of normal activity. Three patients with Sandhoff disease were found to have values ranging from 17% to 37% of normal. These values overlap the range found for carriers of Tay-Sachs disease, and suggest that the usefulness of this substrate, while excellent for diagnosing B variants of GM2 gangliosidosis, requires further study. Perhaps when used together with 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside excellent discriminations of patients, carriers and controls will be realized.

Topics: Acetylglucosaminidase; beta-N-Acetylhexosaminidases; Female; Genetic Carrier Screening; Hexosaminidase A; Hexosaminidases; Humans; Hymecromone; Leukocytes; Male; Sandhoff Disease; Substrate Specificity; Tay-Sachs Disease; Umbelliferones