Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic insect cells
| Autor: | Claire Cahoreau, Yves Combarnous, Sébastien Legardinier, Jean-Claude Poirier, Danièle Klett |
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| Přispěvatelé: | ProdInra, Migration, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2005 |
| Předmět: |
LH
Glycosylation medicine.drug_class Gonadotropins Equine [SDV]Life Sciences [q-bio] Blotting Western Molecular Sequence Data GLYCOSILATION Sf9 [INFO] Computer Science [cs] Biology Spodoptera Biochemistry law.invention 03 medical and health sciences chemistry.chemical_compound Agglutinin N-linked glycosylation law Polysaccharides Lectins medicine Animals [INFO]Computer Science [cs] Horses ComputingMilieux_MISCELLANEOUS Cells Cultured 030304 developmental biology G alpha subunit 0303 health sciences Base Sequence 030302 biochemistry & molecular biology Molecular Mimicry Molecular biology Recombinant Proteins Sialic acid Protein Structure Tertiary [SDV] Life Sciences [q-bio] chemistry Carbohydrate Sequence Cell culture Recombinant DNA Carbohydrate Metabolism Gonadotropin |
| Zdroj: | Glycobiology Glycobiology, Oxford University Press (OUP), 2005, 15 (8), pp.776-790 |
| ISSN: | 0959-6658 1460-2423 |
| Popis: | Recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) was expressed in Mimic 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 Mimic cells. Two types of specific lectin affinity assays (blot analyses and enzyme-linked 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 Siaalpha2,3Gal (based on Maackia amurensis agglutinin [MAA] binding) and Siaalpha2,6Gal (based on Sambucus nigra agglutinin [SNA] binding) were found, but in the alpha subunit dissociated from natural eCG, we only detected Siaalpha2-6Gal. 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 Mimic 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 Galbeta1-3GalNAc structure in recombinant-free alpha and eLH/CG from both Sf9 and Mimic cell lines. Both N- and O-linked carbohydrate side chains synthesized in Mimic cells should thus be amenable to further acellular sialylation. |
| Databáze: | OpenAIRE |
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