Benign HEXA mutations, C739T(R247W) and C745T(R249W), cause beta-hexosaminidase A pseudodeficiency by reducing the alpha-subunit protein levels.

Abstract

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha-subunit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alphaalpha) and Hex A (alphabeta) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta-N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha-subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.

• Authors: Z Cao, E Petroulakis, T Salo, B Triggs-Raine
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 272 Issue 23 Pg. 14975-82 (Jun 06 1997) ISSN: 0021-9258 [Print] United States
• PMID: 9169471 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Isoenzymes
• Macromolecular Substances
• Recombinant Proteins
• Hymecromone
• 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-glucopyranoside
• Hexosaminidase A
• beta-N-Acetylhexosaminidases

Topics

• Adult
• Age of Onset
• Animals
• COS Cells
• Child
• Gangliosidosis, GM1 (enzymology, genetics)
• Hexosaminidase A
• Humans
• Hymecromone (analogs & derivatives, metabolism)
• Infant
• Isoenzymes (genetics, metabolism)
• Kinetics
• Macromolecular Substances
• Point Mutation
• Recombinant Proteins (biosynthesis, metabolism)
• Transfection
• beta-N-Acetylhexosaminidases (deficiency, genetics, metabolism)