[en] The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.
Disciplines :
Chemistry
Author, co-author :
Smargiasso, Nicolas ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Nader, Joseph; Louvain Institute for Biomolecular Science and Technology, UCLouvain, 1348 Louvain-la-Neuve, Belgium.
Mazzucchelli, Gabriel ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Boutry, Marc; Louvain Institute for Biomolecular Science and Technology, UCLouvain, 1348 Louvain-la-Neuve, Belgium.
De Pauw, Edwin ; Université de Liège - ULiège > Département de chimie (sciences)
Chaumont, François ; Louvain Institute for Biomolecular Science and Technology, UCLouvain, 1348 Louvain-la-Neuve, Belgium. francois.chaumont@uclouvain.be.
Navarre, Catherine; Louvain Institute for Biomolecular Science and Technology, UCLouvain, 1348 Louvain-la-Neuve, Belgium.
Language :
English
Title :
Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells.
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