title: A transient tobacco expression system coupled to MALDI-TOF-MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants.
abstract: Tobacco-based transient expression was employed to elucidate the impact of differential targeting to subcellular compartments on activity and quality of gastric lipase as a model for the production of recombinant glycoproteins in plants. Overall N-linked glycan structures of recombinant lipase were analyzed and for the first time sugar structures of its four individual N-glycosylation sites were determined in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on a trypsin digest without isolation or deglycosylation of the peptides. Three glycosylation sites contain both complex-type N-glycans and high-mannose-type structures, the fourth is exclusively linked to high-mannose glycans. Although the overall pattern of glycan structures is influenced by the targeting, our results show that the type of glycans found linked to a given Asn residue is largely influenced by the physico-chemical environment of the site. The transient tobacco system combined with MALDI-TOF-MS appears to be a useful tool for the evaluation of glycoprotein production in plants.
authors.affiliation: MERISTEM Therapeutics, 8 Rue des Frères Lumière, 63100 Clermont-Ferrand, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Mokrzycki-Issartel
authors.forename: Bernadette
authors.initials: B
authors.lastname: Bouchon
authors.forename: Sibille
authors.initials: S
authors.lastname: Farrer
authors.forename: Patricia
authors.initials: P
authors.lastname: Berland
authors.forename: Hélène
authors.initials: H
authors.lastname: Laparra
authors.forename: Jean Claude
authors.initials: JC
authors.lastname: Madelmont
authors.forename: Manfred
authors.initials: M
authors.lastname: Theisen
mesh_terms: D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D004285:Dogs; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D066298:In Vitro Techniques; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000235:genetics*
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D008049:Lipase