Production of human  -hexosaminidase A with highly phosphorylated N-glycans by the overexpression of the Ogataea minuta MNN4 gene

Abstract

Effective enzyme replacement therapy for lysosomal storage diseases requires a recombinant enzyme with highly phosphorylated N-glycans. Recombinant human beta-hexosaminidase A is a potentially therapeutic enzyme for GM2-gangliosidosis. Recombinant HexA has been produced by using the methylotrophic yeast Ogataea minuta as a host, and the purified enzyme was tested for its replacement effect on cultured fibroblasts derived from GM2-gangliosidosis patients. Although the therapeutic effect was observed, in order to obtain the higher therapeutic effect with a little dose as possible, increased phosphorylation of recombinant beta-hexosaminidase A N-glycans is suggested to be prerequisite. In the budding yeast Saccharomyces cerevisiae, the overexpression of MNN4, which encodes a positive regulator of mannosylphosphate transferase, led to increased mannosylphosphate contents. In the present study, we cloned OmMNN4, a homologous gene to ScMNN4, based on the genomic sequence of O. minuta. We overexpressed the cloned gene under the control of the alcohol oxidase promoter in a beta-hexosaminidase A-producing yeast strain. Structural analysis of pyridylamine-labeled N-glycans by high-performance liquid chromatography revealed that the overexpression of MNN4 caused a 3-fold increase in phosphorylated N-glycans of recombinant beta-hexosaminidase A. The recombinant enzyme prepared from strains overexpressing OmMNN4 was more effectively incorporated into cultured fibroblasts and neural cells, and it more rapidly degraded the accumulated GM2-ganglioside as compared to the control enzyme. These results suggest that beta-hexosaminidase A produced in a strain that overexpresses OmMNN4 will act as an effective enzyme for use in replacement therapy of GM2-gangliosidosis.

Human recombinant lysosomal enzymes produced in microorganisms

“…The expression of the recombinant Hex-B was intracellular, but it was directed to the vacuole and was active despite the different glycosylation pattern [79]. Furthermore, production of Hex-A has been reported in the methylotrophic yeast Ogataea minuta [72,80]. In this expression system, the yield of production was 13 mg of Hexo-A per liter.…”

“…Purified Hexo-A showed a final specific activity of 1.4 × 10 6 nmol h −1 mg −1 , which was taken-up by TS or SD fibroblasts in a dose dependent manner after the treatment with α-mannosidase [72]. The insertion of the OmMNN4 gene into O. minuta allowed the production of a recombinant Hexo-A with highly phosphorylated Nglycans (19% in OM4-HexA vs 4.4% in unmodified recombinant Hexo-A), which produced a 10-fold increase in the cellular capture and GM2 degradation [80,81].…”

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis

“…The recombinant human OmHexA and Om4HexA were produced using the strains of Ogataea minuta (Doch1) and prepared by a-mannosidase treatment as previously described. 22,23 The M6P content of the Om4HexA was approximately 3-fold greater than that of OmHexA. On ICV administration, adult Hexb À/À mice were anesthetized with nembutal, and then their hair and skin were removed.…”

Essential Role of Yl MPO1 , a Novel Yarrowia lipolytica Homologue of Saccharomyces cerevisiae MNN4 , in Mannosylphosphorylation of N- and O-Linked Glycans

“…Two Mnn4-like genes exist in S. cerevisiae (19), four exist in P. pastoris (4,27) and Ogataea minuta (1), and eight exist in Candida albicans (17). However, in Y. lipolytica, only a single gene (YALI0D24101g) was predicted to encode a type II membrane protein showing homology with ScMnn4p.…”