Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression.
Plant-specific N-glycosylation, such as the introduction of core alpha1,3-fucose and beta1,2-xylose residues, is a major obstacle to the utilization of plant cell- or plant-derived recombinant therapeutic proteins. The beta1,4-N-acetylglucosaminyltransferase III (GnTIII) introduces a bisecting GlcNAc residue into N-glycans, which exerts a high level of substrate mediated control over subsequent modifications, for example inhibiting mammalian core fucosylation. Based on similar findings in plants, we used Nicotianatabacum BY-2 cells to study the effects of localization and expression levels of GnTIII in the remodeling of the plant N-glycosylation pathway. The N-glycans produced by the cells expressing GnTIII were partially bisected and practically devoid of the paucimannosidic type which is typical for N-glycans produced by wildtype BY-2 suspension cultured cells. The proportion of human-compatible N-glycans devoid of fucose and xylose could be increased from an average of 4% on secreted protein from wildtype cells to as high as 59% in cells expressing chimeric GnTIII, named GnTIII(A.th.) replacing its native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II. The changes in N-glycosylation observed were dependent on the catalytic activity of GnTIII, as the expression of catalytically inactive GnTIII mutants did not show a significant effect on N-glycosylation.
Karg
Saskia R
SR
Institute of Microbiology, ETH Zurich, CH-8093 Zürich, Switzerland. saskia.karg@alumni.ethz.ch
Frey
Alexander D
AD
Kallio
Pauli T
PT
D000818:Animals; D017360:Arabidopsis; D002460:Cell Line; D005643:Fucose / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism; D051381:Rats; D018709:Statistics, Nonparametric; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*
D011506:Proteins; D005643:Fucose; D014994:Xylose; D017350:N-Acetylgalactosaminyltransferases; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase