Production of recombinant beta-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta.
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.
Akeboshi
Hiromi
H
Research Center for Glycoscience, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan.
Chiba
Yasunori
Y
Kasahara
Yoshiko
Y
Takashiba
Minako
M
Takaoka
Yuki
Y
Ohsawa
Mai
M
Tajima
Youichi
Y
Kawashima
Ikuo
I
Tsuji
Daisuke
D
Itoh
Kohji
K
Sakuraba
Hitoshi
H
Jigami
Yoshifumi
Y
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics; D012497:Sandhoff Disease / Q000201:enzymology* / Q000628:therapy; D013661:Tay-Sachs Disease / Q000201:enzymology* / Q000628:therapy; D001619:beta-N-Acetylhexosaminidases / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use
D011994:Recombinant Proteins; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases