Identification of Domains in Human β-Hexosaminidase That Determine Substrate Specificity

The lysosomal β-hexosaminidases are dimers composed of α and β subunits. β-Hexosaminidase A (αβ) is a heterodimer, whereas hexosaminidase B (ββ) and S (αα) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the β-subunit primarily degrades neutral substrates, whereas the α-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only β-hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human β-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both α- and β-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous α-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the β-subunit. Chimeric constructs containing only one of these regions linked to the β-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing α amino acids 1-191 fused with β amino acids 225 to 556, when co-expressed with the wild-type α-subunit, showed activity comparable with that of recombinant β-hexosaminidase A formed by the co-expression of the α- and β-subunits. This result indicates that the β-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of β-hexosaminidase A.