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-subu nit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm t he relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we trans iently expressed each of the benign mutations, and other mutations ass ociated with infantile, juvenile, and adult onset forms of G(M2) gangl iosidosis, as Hex S (alpha alpha) and Hex A (alpha beta) in COS-7 cell s. The benign mutations decreased the expressed Hex A and Hex S activi ty toward the synthetic substrate hylumbelliferyl-6-sulfo-beta-N-acety lglucosaminide (4-MUGS) by 60-80%, indicating that they are the primar y cause of Hex A pseudodeficiency. Western blot analysis showed that t he benign mutations decreased the enzymatic activity by reducing the a lpha-subunit protein level. No change in heat sensitivity, catalytic a ctivity, or the substrate specificity to the synthetic substrates, 4-m ethylumbelliferyl-beta-N-acetylglucosaminide or ylumbelliferyl-6-sulfo -beta-N-acetylglucosaminide, was detected. The effects of the benign m utations on Hex A were further analyzed in fibroblasts, and during tra nsient 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 t he alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our s tudies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expres sion system.