Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in MimicTM insect cells

Recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) was expressed in MimicTM insect cells, that are commercial stably transformed Spodoptera frugiperda (Sf9) cells expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex-type monosialylated N-glycans. We previously showed that it exhibited no in vivo bioactivity although expressing full in vitro bioactivity, and it was suspected that this was because of insufficient sialylation of eLH/CG N-glycans. Lectin binding analyses were performed with recombinant dimeric eLH/ CG or its alpha subunit, secreted in the serum-containing supernatant of infected Sf9 and MimicTM cells. Two types of specific lectin affinity assays (blot analyses and enzymelinked immunosorbent assay) were used to compare the ability or inability of natural and recombinant gonadotropins to bind to various lectins. In natural equine chorionic gonadotropin (eCG), complex-type N-glycans terminating with both Sia 2,3Gal (based on Maackia amurensis agglutinin [MAA] binding) and Sia 2,6Gal (based on Sambucus nigra agglutinin [SNA] binding) were found, but in the alpha subunit dissociated from natural eCG, we only detected Sia 2–6Gal. In eLH/CG and its alpha subunit produced by Sf9 cells, N-glycans were found to be terminated by mannosyl residues (based on Galanthus nivalis agglutinin [GNA] binding), whereas those produced in MimicTM cells were terminated by galactoses (based on binding to Ricinus communis agglutinin I [RCA I] , but not to SNA or MAA). This is in agreement with the fact that the nucleotide donor substrate of sialic acid is not naturally synthesized in insect cells. On the basis of binding to Arachis Hypogaea agglutinin [PNA], O-glycans exhibited the Gal 1–3GalNAc structure in recombinant-free alpha and eLH/ CG from both Sf9 and MimicTM cell lines. Both Nand O-linked carbohydrate side chains synthesized in MimicTM cells should thus be amenable to further acellular sialylation.