MICROHETEROGENEITY OF THE OLIGOSACCHARIDES CARRIED BY THE RECOMBINANTBOVINE LACTOFERRIN EXPRESSED IN MAMESTRA-BRASSICAE CELLS

The development of therapeutic glycoprotein production using the bacul ovirus expression system depends on the ability of insect cell lines t o reproduce site specific mammalian-like N-glycans, A combination of H -1-NMR and mass spectrometry techniques (MALD-MS, ES-MS, and CID-MS-MS ) allowed us to elucidate the N-linked oligosaccharides microheterogen eity on three different N-glycosylation sites, Asn(233), Asn(476), and Asn(545), of a baculovirus-expressed recombinant bovine lactoferrin p roduced in Mamestra brassicae, Two families of N-glycan structures hav e been found: first, oligomannosidic glycans (Man(9-5)GlcNAc(2)) and s econdly, short truncated partially fucosylated glycans (Man(3-2)[Fuc(0 -1)]GlcNAc(2)). These results indicate that Mamestra brassicae cell li ne is not able to synthesize complex N-glycans, even if an alpha 1,6-l inked fucose residue is frequently present on the asparagine-bound N-a cetylglucosamine 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 distr ibution between complex and high-mannose type glycans on the different glycosylation sites, Sites which are naturally occupied by high-manno se glycans (Asn(233) and Asn(545)) are substituted essentially by the same type of N-glycans in the recombinant counterpart, and the site As n476,which carries sialylated complex type chains in the natural glyco protein, is substituted by short, truncated, partially fucosylated cha ins in Mamestra brassicae-expressed bovine lactoferrin, These various results lead us to the conclusion that bovine lactoferrin is an intere sting model to determine the potential of glycosylation of the baculov irus/insect cell expression systems.