| Abstrakt: |
Lysosomal β-hexosaminidase (β-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (αβ) and hexosaminidase B (ββ). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the α subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g.GM2ganglioside. Recently, a point mutation, producing a single amino acid substitution in the α subunit (Arg178-His), has been found to be associated with the Bl variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem.50, 316–318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward α subunit-specific substrates. However, because of the presence of an active β subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected α subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the α and β subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human β subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on β subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that β homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe Bl phenotype results from a totally inactive α-subunit in hexosaminidase A. |
