Human
beta-hexosaminidase A (HexA) is a heterodimeric
glycoprotein composed of alpha- and beta-subunits that degrades GM2
gangliosides in lysosomes.
GM2 gangliosidosis is a
lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2
gangliosides. In order to prepare a large amount of HexA for a treatment based on
enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant
enzymes for ERT. The problem of antigenicity due to differences in N-
glycan structures between mammalian and yeast
glycoproteins was potentially resolved by using alpha-1,6-mannosyltransferase-deficient (och1Delta) yeast as the host. Genes encoding the alpha- and beta-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its
isozymes HexS (alphaalpha) and HexB (betabeta). A total of 57 mg of
beta-hexosaminidase isozymes, of which 13 mg was HexA (alphabeta), was produced per liter of medium. HexA was purified with immobilized
metal affinity column for the His tag attached to the beta-subunit. The purified HexA was treated with
alpha-mannosidase to expose
mannose-6-phosphate (M6P) residues on the N-
glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 +/- 0.1 and 1.7 +/- 0.3 mmol/h/mg, respectively. The
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the beta-subunit of the
recombinant protein. M6PHexA was incorporated dose dependently into
GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated
GM2 ganglioside was observed.