title: Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells.
abstract: The development of therapeutic glycoprotein production using the baculovirus expression system depends on the ability of insect cell lines to reproduce site specific mammalian-like N-glycans. A combination of 1H-NMR and mass spectrometry techniques (MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate the N-linked oligosaccharides microheterogeneity on three different N-glycosylation sites, Asn233, Asn476, and Asn545, of a baculovirus-expressed recombinant bovine lactoferrin produced in Mamestra brassicae. Two families of N-glycan structures have been found: first, oligomannosidic glycans (Man[9-5]GlcNAc2) and secondly, short truncated partially fucosylated glycans (Man(3-2)[Fuc(0-1)]GlcNAc2). These results indicate that Mamestra brassicae cell line is not able to synthesize complex N-glycans, even if an alpha1,6-linked fucose residue is frequently present on the asparagine-bound N-acetylglucosamine residue of short truncated structures. Nevertheless, we have shown that Mamestra brassicae ensures the same N-glycosylation pattern as found on natural bovine lactoferrin showing the same distribution between complex and high-mannose type glycans on the different glycosylation sites. Sites which are naturally occupied by high-mannose glycans (Asn233 and Asn545) are substituted essentially by the same type of N-glycans in the recombinant counterpart, and the site Asn476,which carries sialylated complex type chains in the natural glycoprotein, is substituted by short, truncated, partially fucosylated chains in Mamestra brassicae-expressed bovine lactoferrin. These various results lead us to the conclusion that bovine lactoferrin is an interesting model to determine the potential of glycosylation of the baculovirus/insect cell expression systems.
authors.affiliation: Laboratoire de Chimie Biologique, Unité Mixte de Recherche du CNRS n 111, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
authors.forename: M
authors.initials: M
authors.lastname: Lopez
authors.forename: B
authors.initials: B
authors.lastname: Coddeville
authors.forename: J
authors.initials: J
authors.lastname: Langridge
authors.forename: Y
authors.initials: Y
authors.lastname: Plancke
authors.forename: P
authors.initials: P
authors.lastname: Sautière
authors.forename: H
authors.initials: H
authors.lastname: Chaabihi
authors.forename: F
authors.initials: F
authors.lastname: Chirat
authors.forename: A
authors.initials: A
authors.lastname: Harduin-Lepers
authors.forename: M
authors.initials: M
authors.lastname: Cerutti
authors.forename: A
authors.initials: A
authors.lastname: Verbert
authors.forename: P
authors.initials: P
authors.lastname: Delannoy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D007781:Lactoferrin / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009036:Moths; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D007781:Lactoferrin