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Applied and Environmental Microbiology, August 2007, p. 4805-4812, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00463-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Production of Recombinant ß-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta{triangledown}

Hiromi Akeboshi,1,2 Yasunori Chiba,1,2* Yoshiko Kasahara,1 Minako Takashiba,1 Yuki Takaoka,1 Mai Ohsawa,2,3 Youichi Tajima,2,3 Ikuo Kawashima,2,3 Daisuke Tsuji,2,4 Kohji Itoh,2,4 Hitoshi Sakuraba,2,3 and Yoshifumi Jigami1,2*

Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan,1 CREST, JST, Kawaguchi, Japan,2 Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan,3 Department of Medicinal Biotechnology, Institute for Medicinal Resources, Graduate School of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan4

Received 28 February 2007/ Accepted 24 May 2007

Human ß-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of {alpha}- and ß-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 (och1{Delta}) yeast as the host. Genes encoding the {alpha}- and ß-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS ({alpha}{alpha}) and HexB (ßß). A total of 57 mg of ß-hexosaminidase isozymes, of which 13 mg was HexA ({alpha}ß), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the ß-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 ß-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.

* Corresponding author. Mailing address for Y. Jigami: Research Center for Glycoscience, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Phone: 81-29-861-6160. Fax: 81-29-861-6161. E-mail: jigami.yoshi{at}aist.go.jp. Mailing address for Y. Chiba: Research Center for Glycoscience, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Phone: 81-29- 861-6082. Fax: 81-29-861-6083. E-mail: y-chiba{at}aist.go.jp

{triangledown} Published ahead of print on 8 June 2007.

Applied and Environmental Microbiology, August 2007, p. 4805-4812, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00463-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Akeboshi, H., Kasahara, Y., Tsuji, D., Itoh, K., Sakuraba, H., Chiba, Y., Jigami, Y. (2009). Production of human {beta}-hexosaminidase A with highly phosphorylated N-glycans by the overexpression of the Ogataea minuta MNN4 gene. Glycobiology 19: 1002-1009 [Abstract] [Full Text]  


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