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

• Published in: FEBS letters Vol. 552; no. 2; pp. 170 - 176
• Main Authors: Mokrzycki-Issartel, Nathalie, Bouchon, Bernadette, Farrer, Sibille, Berland, Patricia, Laparra, Hélène, Madelmont, Jean-Claude, Theisen, Manfred
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 25.09.2003
• Subjects: Animals; Binding Sites; Carbohydrate Sequence; Differential targeting; Dogs; Gastric lipase; Gene Expression; Glycoproteins - biosynthesis; Glycoproteins - chemistry; Glycoproteins - genetics; Glycosylation; In Vitro Techniques; Lipase - biosynthesis; Lipase - chemistry; Lipase - genetics; MALDI-TOF-MS; Molecular Sequence Data; Molecular Structure; N-linked glycosylation; Nicotiana - genetics; Plants, Genetically Modified; Polysaccharides - chemistry; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant therapeutic proteins from plants; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transient expression; Gastric lipase; MALDI-TOF-MS; Recombinant therapeutic proteins from plants; N-linked glycosylation; Differential targeting; Transient expression
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(03)00916-5

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.