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.
Fujiyama
K
K
International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565, Japan. fujiyama@icb.osaka-u.ac.jp
Palacpac
N Q
NQ
Sakai
H
H
Kimura
Y
Y
Shinmyo
A
A
Yoshida
T
T
Seki
T
T
D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002460:Cell Line; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000737:chemistry; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011233:Precipitin Tests; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology*
D007527:Isoenzymes; D011134:Polysaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase; D005700:Galactosyltransferases