Glycobiology Advance Access originally published online on June 8, 2009
Glycobiology 2009 19(9):1002-1009; doi:10.1093/glycob/cwp080
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Production of human β-hexosaminidase A with highly phosphorylated N-glycans by the overexpression of the Ogataea minuta MNN4 gene
2 Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566
3 CREST, JST, Kawaguchi 332-0012
4 Department of Medicinal Biotechnology, Institute for Medicinal Resources, Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505
5 Department of Analytical Biochemistry, Meiji Pharmaceutical University, Tokyo 204-8588, Japan
1 To whom correspondence should be addressed: Yasunori Chiba, Tel: +81-29-861-6082; Fax: +81-29-861-6083; e-mail: y-chiba{at}aist.go.jp; Yoshifumi Jigami, Tel: +81-29-861-6160; Fax: +81-29-861-6161; e-mail: jigami.yoshi{at}aist.go.jp
Received on February 25, 2009; revised on April 29, 2009; accepted on June 3, 2009
Effective enzyme replacement therapy for lysosomal storage diseases requires a recombinant enzyme with highly phosphorylated N-glycans. Recombinant human β-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 β-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 β-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 β-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 β-hexosaminidase A produced in a strain that overexpresses OmMNN4 will act as an effective enzyme for use in replacement therapy of GM2-gangliosidosis.
Key words: β-hexosaminidase A / enzyme replacement therapy / GM2-gangliosidosis / methylotrophic yeast / MNN4