Identification of Functional Domains within the α and β Subunits of β-Hexosaminidase A through the Expression of α−β Fusion Proteins

There are three human β-hexosaminidase isozymes which are composed of all possible dimeric combinations of an α and/or a β subunit; A (αβ), B (ββ), and S (αα). The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle > the carboxy-terminal ≫ the amino-terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specificity and thermal stability. The presence of the β subunit in hexosaminidase A also influences the substrate specificity of the α subunit; e.g., in vivo only the A heterodimer can hydrolyze GM2 ganglioside. In this report, we localize functional regions in the two subunits by cellular expression of α/β fusion proteins joined at adjacently aligned residues. First, a chimeric α/β chain was made by replacing the least well-conserved amino-terminal section of the β chain with the corresponding α section. The biochemical characteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical properties. A second fusion protein was made that also included the more homologous middle section of the α chain. This protein expressed the substrate specificity unique to isozymes containing an α subunit (A and S). We conclude that the region responsible for the ability of the α subunit to bind negatively charged substrates is located within residues α132−283. Interestingly, the remaining carboxy-terminal section from the β chain, β316−556, was sufficient to allow this chimera to hydrolyze GM2 ganglioside with 10% the specific activity of heterodimeric hexosaminidase A. Thus, the carboxy-terminal section of each subunit is likely involved in subunit−subunit interactions.