Abstract
The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.
Highlights
There are two major lysosomal beta-hexosaminidase (Hex) isozymes in the normal human tissue; i.e., the highly stable Hex B, a homodimer of beta subunits and the less stable Hex A, a heterodimer composed of beta and alpha subunits
Expression vectors encoding the H1 and H2 Hex hybrids were individually transfected into both an immortalized feline SD fibroblast line and a human infantile TSD Glial cell line [26], and transiently expressed to confirm that their expression would result in increased levels of MUGS hydrolysis (Table 2)
Some TSD cells were transfected with a vector encoding the human WT alpha subunit as a positive control
Summary
There are two major lysosomal beta-hexosaminidase (Hex) isozymes in the normal human tissue; i.e., the highly stable Hex B, a homodimer of beta subunits (encoded by the HEXB gene) and the less stable Hex A, a heterodimer composed of beta and alpha (encoded by the HEXA gene) subunits. The structures confirm previous findings that the ability of the alpha active site to efficiently hydrolyze negatively charged substrates, e.g. MUGS and GM2, comes primarily from two aligned amino acid differences in the subunits, i.e. alpha-N423-. Matsuoka et al used the above information to construct a human hybrid Hex subunit (Table 1) that retained the high stability of the beta subunit while reportedly being able to bind the GM2AP and efficiently hydrolyze GM2 (and MUGS). They suggested that this hybrid Hex could be used for enzyme replacement therapy [12]. Widespread central nervous system (CNS) gene transfer has been demonstrated in feline, porcine, and nonhuman primate animal models [17,18,19,20,21], suggesting the possibil-
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