Isolation of the plant mutants involved in N-glycosylation pathway and their use for humanized N-glycosylation

N-glycosylation is a major post-translational protein modification, which alters physicochemical properties of the protein, affecting the folding, distribution, stability and thus biological function and efficiency of protein. Plant type complex N-glycans are distinctive from those found in mammalian because they contain β1,2-xylose and core α 1,3-fucose residues attached to the pentasaccharide (Man3GlcNAc2) core structure but no sialic acid residues. The presence of β1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans has long been an irritating limitation in the use of plant-made pharmaceuticals (PMPs) in human therapy, as these N-glycan epitopes are potentially immunogenic in mammals. In this study, to remove the plant specific sugar residues and humanize the N-glycosylation in plant, we isolated mutants of the corresponding plant specific glycosyltransferase genes (α1,3-fucosyltransferase I, α1,3-fucosyltransferase II, β 1,2-xylosyltransferase, β1,3-galactosyltransferase and α1,4-fucosyltransferase). We made double, triple and quadruple mutants by crossing the mutants, and quintuple mutant is on the construction. The triple (fuct1/fuct2/xylt) and quadruple (fuct1/fuct2/xylt/fuct3) mutants do not show significant developmental defects in a normal growth condition and they do not produce the plant specific sugar residues on the N-glycan. The resulting mutant will be transformed by human α1,6-fucosyltransferase and β1,4-galactosyltransferase genes to accomplish further humanized N-glycosylation in plant. [Supported by BK21 program]