Publications

• Direct determination of the substrate specificity of the alpha-active site in heterodimeric beta-hexosaminidase A.

  Hou Y., Tse R., Mahuran D.J.

  The beta-hexosaminidase isozymes are produced through the combination of alpha and beta subunits to form any one of three active dimers (monomeric subunits are not functional). Heterodimeric hexosaminidase A (alpha beta) is the only isozyme that can hydrolyze GM2 ganglioside in vivo, requiring the ... >> More

  The beta-hexosaminidase isozymes are produced through the combination of alpha and beta subunits to form any one of three active dimers (monomeric subunits are not functional). Heterodimeric hexosaminidase A (alpha beta) is the only isozyme that can hydrolyze GM2 ganglioside in vivo, requiring the presence of the GM2 activator protein. Hexosaminidase S (alpha alpha) exists but is not considered a physiological isozyme. Although hexosaminidase B (beta beta) is present in normal human tissues, it has no known unique function in vivo. However, a unique function for the beta-active site present in both hexosaminidase A and B has been indicated in a previous study of the various substrate specificities of the homodimeric forms of hexosaminidase (S and B). It was concluded that the alpha-active site is only able to efficiently hydrolyze negatively charged substrates, and the beta-active site is only able to hydrolyze neutral substrates. When this model of nonoverlapping alpha- and beta-substrates is extrapolated to heterodimeric hexosaminidase A, it has a major effect on the interpretation of recent results relating to the mode of action of the GM2 activator protein. In this report, we directly examine these substrate specificities using a novel form of hexosaminidase A containing an inactive beta subunit, produced in permanently transfected CHO cells. We demonstrate that, whereas the beta-active site has the same substrate specificities in either its A-heterodimeric or B-homodimeric forms, the alpha-active site in the A-heterodimer has different kinetic parameters than the alpha-active site in the S-homodimer. We conclude that the alpha and beta subunits in hexosaminidase A participate equally in the hydrolysis of neutral substrates. << Less

  Biochemistry 35:3963-3969(1996) [PubMed] [EuropePMC]

• Classification of disorders of GM2 ganglioside hydrolysis using 3H-GM2 as substrate.

  Novak A., Callahan J.W., Lowden J.A.

  Rates of GM2 ganglioside hydrolysis by fibroblasts from normal controls and patients with GM2 gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decrease in cell-incorporated 3H-GM2 over time, and in vitro by assaying the rate of 3H-GM2 hydrolysis using fibr ... >> More

  Rates of GM2 ganglioside hydrolysis by fibroblasts from normal controls and patients with GM2 gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decrease in cell-incorporated 3H-GM2 over time, and in vitro by assaying the rate of 3H-GM2 hydrolysis using fibroblast extracts in the presence of no additives, sodium taurocholate, and GM2 activator protein. In tissue culture, normal cells hydrolyzed cell-incorporated GM2 while fibroblasts from patients with GM2 gangliosidosis did not. The half life of GM2 in normal fibroblasts was 78 hours. In vitro, only normal fibroblast extracts hydrolyzed GM2 in the absence of additives. In the presence of 10 mM sodium taurocholate, rates of GM2 hydrolysis by normal fibroblast extracts were increased 5-16-fold, fibroblast extracts from AB and B1 variant patients hydrolyzed GM2 at normal rates, cell extracts from patients with Tay-Sachs disease hydrolyzed GM2 at nearly normal rates, and cell extracts from Sandhoff disease patients hydrolyzed GM2 at about 10% of normal rates. In the presence of 1 microgram of GM2 activator, rates of GM2 hydrolysis by normal fibroblast extracts were increased 8-25-fold, fibroblast extracts from a patient with the AB variant hydrolyzed GM2 at normal rates, and cell extracts from other variants of GM2 gangliosidosis did not hydrolyze GM2. The results suggest that measuring the persistence of 3H-GM2 in tissue culture over time will detect any variant of GM2 gangliosidosis and may be the ideal way to test for the presence of this disease. Variants can be distinguished by assaying the hydrolysis of 3H-GM2 using cell extracts in the absence of additives, with sodium taurocholate, and with activator. << Less

  Biochim. Biophys. Acta 1199:215-223(1994) [PubMed] [EuropePMC]

• A Pro504 --> Ser substitution in the beta-subunit of beta-hexosaminidase A inhibits alpha-subunit hydrolysis of GM2 ganglioside, resulting in chronic Sandhoff disease.

  Hou Y., McInnes B., Hinek A., Karpati G., Mahuran D.

  The GM2 gangliosidoses are caused by mutations in the genes encoding the alpha-(Tay-Sachs) or beta-(Sandhoff) subunits of heterodimeric beta-hexosaminidase A (Hex A), or the GM2 activator protein (AB variant), a substrate-specific co-factor for Hex A. Although the active site associated with the h ... >> More

  The GM2 gangliosidoses are caused by mutations in the genes encoding the alpha-(Tay-Sachs) or beta-(Sandhoff) subunits of heterodimeric beta-hexosaminidase A (Hex A), or the GM2 activator protein (AB variant), a substrate-specific co-factor for Hex A. Although the active site associated with the hydrolysis of GM2 ganglioside, as well as part of the binding site for the ganglioside-activator complex, is associated with the alpha-subunit, elements of the beta-subunit are also involved. Missense mutations in these genes normally result in the mutant protein being retained in the endoplasmic reticulum and degraded. The mutations associated with the B1-variant of Tay-Sachs are rare exceptions that directly affect residues in the alpha-active site. We have previously reported two sisters with chronic Sandhoff disease who were heterozygous for the common HEXB deletion allele. Cells from these patients had higher than expected levels of mature beta-protein and residual Hex A activity, approximately 20%. We now identify these patients' second mutant allele as a C1510T transition encoding a beta-Pro504 --> Ser substitution. Biochemical characterization of Hex A from both patient cells and cotransfected CHO cells demonstrated that this substitution (a) decreases the level of heterodimer transport out of the endoplasmic reticulum by approximately 45%, (b) lowers its heat stability, (c) does not affect its Km for neutral or charged artificial substrates, and (d) lowers the ratio of units of ganglioside/units of artificial substrate hydrolyzed by a factor of 3. We concluded that the beta-Pro504 --> Ser mutation directly affects the ability of Hex A to hydrolyze its natural substrate but not its artificial substrates. The effect of the mutation on ganglioside hydrolysis, combined with its effect on intracellular transport, produces chronic Sandhoff disease. << Less

  J. Biol. Chem. 273:21386-21392(1998) [PubMed] [EuropePMC]