Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells.

Human CMP-N-acetylneuraminic acid (NeuAc) synthase (hCSS) and α2,6-sialyltransferase (hST) participate in the sialylation of N-linked glycans in mammalian cells. hCSS synthesizes CMP-NeuAc, which hST uses as a donor substrate to transfer NeuAc to the terminal position of N-linked glycans. In plant cells, the presence of NeuAc has not yet been substantiated and the identification of the genes involved in the sialylation of N-glycan has not been carried out. In this study, we introduced hCSS and hST genes into suspension-cultured tobacco BY2 cells to provide the machinery for the sialylation pathway in plants. hCSS and hST stably expressed in the plant cells showed activity. In addition, CMP-NeuAc produced by hCSS in the transformed plant cells functioned as a donor substrate to hST. An in vitro coupled hCSS and hST reaction resulted in the production of mammalian-type sialoglycoproteins bearing terminal NeuAc residues. Furthermore, the results of the purification of the coupled-reaction products by Sambucus sieboldian lectin column chromatography and digestion with linkage-specific neuraminidase revealed that the modified terminal residue was α2,6-linked NeuAc. Here, we demonstrate that the in vitro sialylation of N-linked glycans on mammalian proteins can be achieved using plant cell extracts stably expressing hCSS and hST, providing proof-of-principle that a sialylated human therapeutic protein can be produced in plants.

Kajiura H ，Misaki R ，Fujiyama K ，Seki T ... -  《-》

Reversible sialylation: synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid from cytidine 5'-monophosphate with alpha2,3-sialyl O-glycan-, glycolipid-, and macromolecule-based donors yields diverse sialylated products.

Chandrasekaran EV ，Xue J ，Xia J ，Locke RD ，Matta KL ，Neelamegham S ... -  《-》

Probing the CMP-Sialic Acid Donor Specificity of Two Human β-d-Galactoside Sialyltransferases (ST3Gal I and ST6Gal I) Selectively Acting on O- and N-Glycosylproteins.

Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio-pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP-sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human β-d-galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP-sialic acids: CMP-Neu5Ac, and CMP-Neu5N-(4pentynoyl)neuraminic acid (CMP-SiaNAl), an unnatural CMP-sialic acid donor with an extended and functionalized N-acyl moiety.

Noel M ，Gilormini PA ，Cogez V ，Yamakawa N ，Vicogne D ，Lion C ，Biot C ，Guérardel Y ，Harduin-Lepers A ... -  《-》

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

Misaki R ，Fujiyama K ，Seki T 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Ecto-sialyltransferase of human B lymphocytes reconstitutes differentiation markers in the presence of exogenous CMP-N-acetyl neuraminic acid.

Gross HJ ，Merling A ，Moldenhauer G ，Schwartz-Albiez R ... -  《BLOOD》