title: Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells.
abstract: 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.
authors.affiliation: International Center for Biotechnology, Osaka University, Suita-shi, Osaka 565-0871, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kajiura
authors.forename: Ryo
authors.initials: R
authors.lastname: Misaki
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Seki
mesh_terms: D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D000216:N-Acylneuraminate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase