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

• Published in: Glycobiology (Oxford) 2009-09, Vol.19 (9), p.1002-1009
• Main Authors: Akeboshi, Hiromi, Kasahara, Yoshiko, Tsuji, Daisuke, Itoh, Kohji, Sakuraba, Hitoshi, Chiba, Yasunori, Jigami, Yoshifumi
• Format: Article
• Language: English
• Subjects: Saccharomyces cerevisiae
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwp080

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.