Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize.
Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.
Bondili
Jayakumar Singh
JS
Institute of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, BOKU-Vienna, Muthgasse 18, A-1190 Vienna, Austria.
Castilho
Alexandra
A
Mach
Lukas
L
Glössl
Josef
J
Steinkellner
Herta
H
Altmann
Friedrich
F
Strasser
Richard
R
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018745:Genome, Plant; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D018411:Spodoptera / Q000235:genetics; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D010430:Pentosyltransferases; C408586:beta 1,2-xylosyltransferase