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Glycobiology Advance Access published online on April 6, 2005
Glycobiology, doi:10.1093/glycob/cwi060
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Received November 3, 2004
Revised March 29, 2005
Accepted March 29, 2005

Article

Mammalian-Like Non-Sialyl Complex-Type N-Glycosylation of Equine Gonadotropins in MimicTM Insect Cells

Sébastien Legardinier 1, Danièle Klett 1, Jean-Claude Poirier 1, Yves Combarnous 1, and Claire Cahoreau 1*

1 Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS) et Université François Rabelais de Tours, 37 380 Nouzilly, France.

* To whom correspondence should be addressed.
Claire Cahoreau, E-mail: cahoreau{at}tours.inra.fr


  Abstract

Recombinant equine Luteinizing Hormone/Chorionic Gonadotropin (eLH/CG) was expressed in MimicTM insect cells, that are commercial stably transformed 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 due to 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 ELISA) were used to compare the ability or inability of natural and recombinant gonadotropins to bind to various lectins.

In natural eCG, complex-type N-glycans terminating with both Siaa2,3Gal (based on Maackia amurensis agglutinin (MAA) binding) and Sia{alpha}2,6Gal (based on Sambucus nigra agglutinin (SNA) binding) were found but in the alpha subunit dissociated from natural eCG, we only detected Sia{alpha}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, 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.

Based on binding to Arachis Hypogaea agglutinin (PNA), O-glycans exhibited the Gal{beta}1-3GalNac structure in recombinant free alpha and eLH/CG from both Sf9 and MimicTM cell lines. Both N- and O-linked carbohydrate side chains synthesized in MimicTM cells should thus be amenable to further acellular sialylation.

Keywords: Baculovirus /gonadotropin/glycosylation /insect cells /lectin.
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 J Mol EndocrinolHome page
S. Legardinier, J.-C. Poirier, D. Klett, Y. Combarnous, and C. Cahoreau
Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants
J. Mol. Endocrinol., April 1, 2008; 40(4): 185 - 198.
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