Biochemical characterization of the Cys138Arg substitution associated with the AB variant form of GM2 gangliosidosis: evidence that Cys138 is required for the recognition of the GM2 activator/GM2 ganglioside complex by beta-hexosaminidase A.
The function of the GM2 activator protein is to act as a substrate-specific cofactor in the hydrolysis of GM2 ganglioside by beta-hexosaminidase A. Mutations in the gene encoding it result in the AB variant form of GM2 gangliosidosis. One such mutation, Cys138 Arg, results in the mutant protein being retained and degraded in the endoplasmic reticulum of mammalian cells. In order to characterize the biochemical effects of this substitution, we expressed the mutant protein in transformed bacteria. We first compared the wild-type protein produced by two bacterial expression methods, one requiring protein refolding, with activator purified from the medium of transfected CHO cells. The "activity" and circular dichroism spectrum (alpha-helical content) of all three proteins were similar, justifying the use of refolded activator from transformed bacteria in structure/function studies. Second, the mutant protein was expressed in both bacterial systems and in each retained approximately 2% of the wild type's specific activity. The presence of even this small amount of activity in the mutant protein coupled with a calculated alpha-helical content nearly identical to the wild type, strongly suggest that no major tertiary or secondary structural changes, respectively, had occurred due to the mutation. However, we demonstrate that its heat stability at 60 degrees C is reduced 14-fold, suggesting some localized change in tertiary structure. The loss of a disulfide loop was confirmed by reacting the mutant protein with Ellman's reagent. A kinetic analysis detected a large increase in the apparent K(m) of beta-hexosaminidase A for the mutant; however, there was no apparent change in Vmax. A fluorescence dequenching assay was used to evaluate the ability of the mutant protein to transport lipids and bind GM2 ganglioside. These assays detected no difference between the wild-type and mutant proteins, indicating that the Cys138 Arg substitution has no effect on these functions. We conclude that the mutation specifically affects a domain in the activator protein that is responsible for the recognition of the activator-GM2 ganglioside complex by beta-hexosaminidase A.