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.
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.
Mokrzycki-Issartel
Nathalie
N
MERISTEM Therapeutics, 8 Rue des Frères Lumière, 63100 Clermont-Ferrand, France.
Bouchon
Bernadette
B
Farrer
Sibille
S
Berland
Patricia
P
Laparra
Hélène
H
Madelmont
Jean Claude
JC
Theisen
Manfred
M
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*
D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D008049:Lipase