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Efficient uptake of recombinant ?-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys.

ABSTRACT: To economically produce recombinant human ?-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes ?-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.

SUBMITTER: Tsukimura T

PROVIDER: S-EPMC3269645 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Efficient uptake of recombinant α-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys.

Tsukimura Takahiro T Kawashima Ikuo I Togawa Tadayasu T Kodama Takashi T Suzuki Toshihiro T Watanabe Toru T Chiba Yasunori Y Jigami Yoshifumi Y Fukushige Tomoko T Kanekura Takuro T Sakuraba Hitoshi H

Molecular medicine (Cambridge, Mass.) 20120210

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the reco ...[more]

PMID: 22033676

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Project description:Fabry disease is an X-linked genetic disorder characterized by deficient activity of ?-galactosidase A (GLA) and accumulation of glycolipids, and various GLA gene mutations lead to a wide range of clinical phenotypes from the classic form to the later-onset one. To investigate the biochemical heterogeneity and elucidate the basis of the disease using available clinical samples, we measured GLA activity, GLA protein and accumulated globotriaosylsphingosine (Lyso-Gb3), a biomarker of this disease, in plasma samples from Fabry patients. The analysis revealed that both the enzyme activity and the protein level were apparently decreased, and the enzyme activity was well correlated with the protein level in many Fabry patients. In these cases, a defect of biosynthesis or excessive degradation of mutant GLAs should be involved in the pathogenesis, and the residual protein level would determine the accumulation of Lyso-Gb3 and the severity of the disease. However, there are some exceptional cases, i.e., ones harboring p.C142Y, p.R112H and p.M296I, who exhibit a considerable amount of GLA protein. Especially, a subset of Fabry patients with p.R112H or p.M296I has been attracted interest because the patients exhibit almost normal plasma Lyso-Gb3 concentration. Structural analysis revealed that C142Y causes a structural change at the entrance of the active site. It will lead to a complete enzyme activity deficiency, resulting in a high level of plasma Lyso-Gb3 and the classic Fabry disease. On the other hand, it is thought that R112H causes a relatively large structural change on the molecular surface, and M296I a small one in a restricted region from the core to the surface, both the structural changes being far from the active site. These changes will cause not only partial degradation but also degeneration of the mutant GLA proteins, and the degenerated enzymes exhibiting small and residual activity remain and probably facilitate degradation of Lyso-Gb3 in plasma, leading to the later-onset phenotype. The results of this comprehensive analysis will be useful for elucidation of the basis of Fabry disease.

Dataset Information

Efficient uptake of recombinant ?-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys.

Publications

Efficient uptake of recombinant α-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys.

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