Benign HEXA mutations, C739T(R247W) and C745T(R249W), cause beta-hexosaminidase A pseudodeficiency by reducing the alpha-subunit protein levels.
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.
Cao
Z
Z
Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada.
Petroulakis
E
E
Salo
T
T
Triggs-Raine
B
B
D000328:Adult; D017668:Age of Onset; D000818:Animals; D019556:COS Cells; D002648:Child; D016537:Gangliosidosis, GM1 / Q000201:enzymology / Q000235:genetics; D054818:Hexosaminidase A; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007223:Infant; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism
D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006923:Hymecromone; C043647:4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-glucopyranoside; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases