Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell

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Abstract

Plant cells have no β1,4-galactosylated and sialylated glycan, which plays important roles in biological functions in animal cells. Previously, we generated transgenic tobacco BY2 suspension-cultured cells that produced human β1,4-galactosyltransferase [N.Q. Palacpac, S. Yoshida, H. Sakai, Y. Kimura, K. Fujiyama, T. Yoshida, T. Seki, Stable expression of human β1,4-galactosyltransferase in plant cells modifies N-linked glycosylation pattern, Proc. Natl. Acad. Sci. USA 96 (1999) 4692–4697]. In this study, we introduced two critical genes encoding human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter into tobacco suspension-cultured cell to pave a route for sialic biosynthetic pathway. The recombinant human proteins showed their biological activities. These results show that the plant cell can be a useful bioreactor for the production of mammalian glycoproteins.

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Materials and methods

Cloning of hCSS and hCST gene, and construction of the plant expression vector. hCSS and hCST gene were amplified by PCR. The gene for hCSS was amplified from human kidney cDNA (Clontech Laboratories, USA) by PCR using primers hCSS-P1 (5′-GTTACTAGTATGGACTCGGTGGAGAAGGGGGCCGCCACCTCCGTCTCCAACCCGCGGGGGCGACCGTCCC-3′) and hCSS-P2 (5′-TGGGAGCTCCTATTTTTGGCATGAATTATT-3′). For hCST gene, primers hCST-P1 (5′-GTTAGATCTATGGCTGCCCCGAGAGACAAT-3′) and hCST-P2 (5′-TTGGAGCTCTCACACACCAATAACTCTCTC-3′) were used to

Analysis of tobacco suspension-cultured cells expressing hCSS

In vitro synthesis of CMP-sialic acid was carried out using the extracts of transformant hCSS15 and wild-type BY2. The reaction products were transferred onto PA sugar chain (Gal2GN2M3-PA; β-d-Gal-(1  4)-β-d-GlcNAc-(1  2)-α-d-Man-(1  6)[β-d-Gal-(1  4)-β-d-GlcNAc-(1  2)-α-d-Man-(1  3)]β-d-Man-(1  4)-β-d-GlcNAc-(1  4)-GlcNAc-PA) under the human α2,6-sialyltransferase, and then the resulting products were analyzed by HPLC apparatus. Elution positions of transferred PA sugar chain corresponded to those of

Discussion

Glycoproteins of pharmaceutical importance have been produced in plant as bioreactor. Because sialic acid plays a lot of physiological roles, production of glycoproteins with sialic acids in plant should be ideal systems. However, the synthetic pathway of sialic acid in plant cells has not been cleared although Shah et al. reported. In this study, we succeeded in expressing both gene of human CMP-sialic acid synthetase and human CMP-sialic acid transporter gene in tobacco BY2

Acknowledgments

We thank Dr. Kazuhisa Aoki, Dr. Nobuhiro Ishida, and Dr. Masao Kawakita for helpful discussions.

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