Production of recombinant beta-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta

Abstract

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.

FIG. 1.

Analysis of HexA and M6PHexA by SDS-PAGE (A) and Western blotting (B). Purified HexA and M6PHexA were separated by 10% SDS-PAGE and analyzed by CBB staining (A) and immunostaining (B). The blotted membrane was overlaid with rabbit anti-human HexA serum, followed by alkaline phosphatase-conjugated anti-rabbit immunoglobulin G as the secondary antibody. CDP-Star detection reagent (GE Healthcare Bio-Sciences Corp.) was used to visualize the enzymes. M, molecular marker; lane 1, HexA; lane 2, M6PHexA.

FIG. 2.

Primary structure and inner processing sites of the α- and β-subunits of human HexA. (A) EndoH_f-treated HexA blotted onto PVDF membrane and stained with CBB. The EndoH_f treatment was performed according to the manufacturer's instructions. The two major bands (α and β) were analyzed for their N-terminal sequences. The primary structures of the α-subunit (B) and the β-subunit (C) of HexA are shown. Asterisks show the N-terminal amino acid detected by protein sequencing. Signal sequences are shown by the arrows. Amino acids that are proteolytically processed in mammalian HexA are boxed.

FIG. 3.

GM2 ganglioside hydrolysis by recombinant HexA. Applied samples are as follows: 1, GM2 standard (GM2 1.2 μg plus sodium taurodeoxycholate [TD], 30 μg); 2, GM2 plus TD plus human placenta β-hexosaminidase for a 3-h incubation; 3, GM2 plus TD plus human placenta β-hexosaminidase for a 24-h incubation; 4, GM2 plus TD plus HexA for a 1-h incubation; 5, GM2 plus TD plus HexA for a 3-h incubation; 6, GM2 plus TD plus HexA for a 24-h incubation. The enzymatic reaction product by recombinant HexA produced in O. minuta was identified as GM3 based on the R_f value on TLC, which was identical with that of the authentic human placenta-derived β-hexosaminidase enzyme.

FIG. 4.

Interaction between Dom9His and M6PHexA. Blotted recombinant enzymes were detected by anti-HexA antibody (A) and Dom9His (B), as described in Materials and Methods. Lane 1, HexA; lane 2, M6PHexA.

FIG. 5.

Analysis of M6PHexA uptake by TS and SD fibroblasts. (A) TS and SD fibroblasts were cultured in medium containing recombinant HexA enzymes (600 nmol/h/well, toward MUGS), and the MUGS-hydrolyzing activity of homogenates was measured to determine enzyme incorporation. 1, no enzyme addition; 2, HexA; 3, M6PHexA; 4, M6PHexA with 5 mM M6P. Each bar represents the mean of two independent experiments. The cellular MUGS-hydrolyzing activity of fibroblasts from a normal subject was 752 nmol/h/mg. Error bars represent standard errors of the means. (B) Dose dependency of enzyme uptake was determined by the addition of M6PHexA to the TS and SD fibroblasts at the MUGS-hydrolyzing activities of 200, 600, and 1,800 nmol/h/well. The MUGS-hydrolyzing activity of cell extracts was determined to detect enzyme incorporation.

FIG. 6.

Analysis of intracellular GM2 ganglioside degradation in TS fibroblasts. After the TS fibroblasts were cultured in medium that contained various amounts of recombinant enzymes, they were fixed and intracellular GM2 gangliosides were detected by antibody to GM2 ganglioside. (A) TS fibroblasts; (B) TS fibroblasts plus M6PHexA (600 nmol/h/well); (C) TS fibroblasts plus M6PHexA (1,800 nmol/h/well); (D) normal fibroblasts. Bar, 10 μm.