Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells

Abstract

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.

Inactivation of N-Acetylglucosaminyltransferase I and α1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans

“…For the mass spectrometry analysis, cultures were grown in liquid D11b medium ( Vasilev et al, 2013 ) without cyclodextrin for 10 days in 50 mL of medium in 250 mL Erlenmeyer flasks. Extracellular proteins were collected by filtration on three layers of Miracloth (Calbiochem), then centrifuged at 8,000 g for 30 min, and precipitated by salting-out as previously described in Smargiasso et al (2019) with the following minor adaptations: solid (NH 4 ) 2 SO 4 was slowly added to the culture medium with stirring at room temperature to 55% saturation and the solution was then kept for 2 h at 4°C and centrifuged at 3,000 g for 40 min at 4°C. The pellet was solubilized in 140 μL of PBS buffer and desalted on a PD-10 filtration column (GE Healthcare, Uppsala, Sweden) equilibrated with PBS buffer.…”

“…N. tabacum Bright Yellow-2 (BY-2) suspension cells are characterized by a short culture cycle, easy scaling up in bioreactors, low risk of contamination by human pathogens and the possibility to purify secreted recombinant proteins directly from the culture medium ( Tekoah et al, 2015 ; Santos et al, 2016 ). BY-2 cells have already been used to express several pharmaceutical glycoproteins such as antibodies ( De Muynck et al, 2009 ; Holland et al, 2010 ; Magy et al, 2014 ), a viral glycoprotein ( Smargiasso et al, 2019 ) or human enzymes ( Hanania et al, 2017 ; Ilan et al, 2017 ). In this study, we report on the identification and characterization of two GnTI genes in the N. tabacum BY-2 genome.…”

Glycomics and glycoproteomics of viruses: Mass spectrometry applications and insights toward structure–function relationships

“…Smargiasso et al (2019) studied human cytomegalovirus glycoprotein B grown in different cell lines. They used an in‐house program to search MS 2 HCD data for oxonium and HexNAc + peptide ions, then manually curated the glycosyl‐containing MS 2 spectra.…”

Mass Spectrometry-Based Structural Virology

“…Such combined fragmentation methods to identify viral glycopeptides were applied in the bulk of pioneering studies on HIV Env glycosylation, 115,129,143,144,146,151 for the characterization of the N-glycans decorating glycoprotein B of cytomegalovirus and E2 of Hepatitis C virus. 121,131 In the case of glycosylation characterization of heamagglutinin-neuraminidase from Newcastle disease virus, a fragmentation strategy was implemented wherein MS/MS spectra were acquired with HCD fragmentation, which upon identification of oxonium ions triggered either ETD or CID fragmentation of the same precursor, a technique called HCD-pd-ETD(CID), which boosts specifically the numbers of identified glycopeptide fragmentation spectra and improves the glycopeptide assignments. 134 In recent years, a hybrid fragmentation strategy known as EThcD (electron transfer/higher energy collision-induced dissociation), in which peptides are fragmented with ETD and subsequent supplemental HCD activation, 156 was shown to be beneficial for the characterization of glycopeptides and had a positive effect on the field of viral glycoproteomics.…”