In vivo conversion of a glycan to human compatible type by transformed tobacco cells.

Horseradish peroxidase isozyme C (HRP; EC 1.11.1.7) was used as a model protein to evaluate the capacity of tobacco cells transformed with human beta 1,4-galactosyltransferase (GT6) to modify and galactosylate a foreign glycoprotein. Cells transformed with the HRP gene are designated as BY2-HRP and GT6-HRP, for wild type BY2 and GT6 transformed cells, respectively. Expression of HRP cells was confirmed by isoelectric focusing, peroxidase activity staining, Western blotting, and enzymatic assays. The presence of HRP galactosylated N-glycans in GT6-HRP cells was analyzed by lectin staining, affinity chromatography, and structural analyses of pyridylamino-labeled RCA(120)-bound sugar chains. The structure of Gal(1)GlcNAc(1)Man(5)GlcNAc(2) was proposed based from the results of exoglycosidase digestions and two-dimensional sugar chain mapping. Unlike the HRP produced in BY2-HRP cells, the HRP from GT6-HRP cells has galactosylated glycoproteins that did not bind to the xylose-specific antiserum, suggesting the absence of the beta 1,2-xylose residue in the sugar chain.

Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns.

beta1,4-Galactosyltransferase (UDP galactose: beta-N-acetylglucosaminide: beta1,4-galactosyltransferase; EC 2.4.1. 22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blotting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligosaccharide structures shows that the galactosylated N-glycans account for 47.3% of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the transformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glycoproteins.

Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells.

The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1 Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3 Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that alpha(1-->3) fucosylation of N-glycans does occur but beta(1-->4) galactosylation of the sugar chains does not in the tobacco cultured cells.