Abstract
Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N-glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N-glycosylation in alfalfa. The first was a knock-down of two plant-specific N-glycan maturation enzymes, beta1,2-xylosyltransferase and alpha1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human beta1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of alpha1,3-fucose- and beta1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human beta1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Cell Line
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Cloning, Molecular
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Down-Regulation*
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Epitopes / genetics*
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Epitopes / immunology
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Fucosyltransferases / antagonists & inhibitors
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Fucosyltransferases / chemistry
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Fucosyltransferases / genetics
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Galactosyltransferases / genetics*
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Galactosyltransferases / metabolism
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Glycoproteins / chemistry
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Glycoproteins / genetics
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Glycoproteins / metabolism
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Glycosylation
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Humans
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Medicago sativa / genetics*
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Medicago sativa / metabolism
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Molecular Sequence Data
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N-Acetylglucosaminyltransferases / chemistry
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N-Acetylglucosaminyltransferases / genetics
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N-Acetylglucosaminyltransferases / metabolism
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Nicotiana / genetics
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Pentosyltransferases / antagonists & inhibitors
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Pentosyltransferases / chemistry
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Pentosyltransferases / genetics
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Plant Proteins / antagonists & inhibitors
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Plant Proteins / genetics
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Plant Proteins / metabolism
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Plants, Genetically Modified / metabolism
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RNA Interference
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Recombinant Fusion Proteins / metabolism
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Sequence Alignment
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Spodoptera
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Substrate Specificity
Substances
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Epitopes
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Glycoproteins
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Plant Proteins
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Recombinant Fusion Proteins
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Fucosyltransferases
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Galactosyltransferases
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N-Acetylglucosaminyltransferases
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beta1,4-galactosyltransferase, human
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alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I
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galactoside 3-fucosyltransferase
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Pentosyltransferases
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beta 1,2-xylosyltransferase