Abstract
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 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.
Highlights
Fabry disease (MIM 301500) is an X-linked inborn error of metabolism with a high incidence of 1 in 1,250–4,000 male live births [1,2,3]
Two different recombinant human galactosidase A (GLA), agalsidase alfa (Replagal®; Shire HGT, Cambridge, MA, USA) [8], produced with cultured human fibroblasts, and agalsidase beta (Fabrazyme®; Genzyme, Cambridge, MA, USA) [9,10], generated by Chinese hamster ovary (CHO) cells, have been developed, and they are used for enzyme replacement therapy (ERT) for Fabry disease
Having mammalianlike, phosphomannosylated sugar chains, we expressed human GLA in a gene-manipulated O. minuta strain with the OCH1 gene, which encodes α-1,6-mannosyltransferase catalyzing the initial reaction of the outer sugar chain synthesis specific to yeast, deleted and overexpressing the S. cerevisiae MNN4 gene, which encodes a positive regulator of mannosylphosphate transferase
Summary
Fabry disease (MIM 301500) is an X-linked inborn error of metabolism with a high incidence of 1 in 1,250–4,000 male live births [1,2,3]. Two different recombinant human GLAs, agalsidase alfa (Replagal®; Shire HGT, Cambridge, MA, USA) [8], produced with cultured human fibroblasts, and agalsidase beta (Fabrazyme®; Genzyme, Cambridge, MA, USA) [9,10], generated by Chinese hamster ovary (CHO) cells, have been developed, and they are used for enzyme replacement therapy (ERT) for Fabry disease. These recombinant GLAs are glycoproteins having both complex type and highmannose type sugar chains produced by cultured mammalian cells, and it is thought that they are incorporated into cells mainly via mannose 6-phosphate (M6P) receptors in many organs [11] except for the liver, in which uptake by hepatocytes and Kupffer cells occurs mainly through asialoglycoprotein receptors and mannose ones, respectively [12].
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