Identification of N-glycans from Ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry

Abstract

The larger fragment of the transmembrane glycoprotein (GP1) and the soluble glycoprotein (sGP) of Ebola virus were expressed in human embryonic kidney cells and the secreted products were purified from the supernatant for carbohydrate analysis. The N-glycans were released with PNGase F from within sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) gels. Identification of the glycans was made with normal-phase high-performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionisation mass spectrometry, negative ion electrospray ionisation fragmentation mass spectrometry and exoglycosidase digestion. Most glycans were complex bi-, tri- and tetra-antennary compounds with reduced amounts of galactose. No bisected compounds were detected. Triantennary glycans were branched on the 6-antenna; fucose was attached to the core GlcNAc residue. Sialylated glycans were present on sGP but were largely absent from GP1, the larger fragment of the transmembrane glycoprotein. Consistent with this was the generally higher level of processing of carbohydrates found on sGP as evidenced by a higher percentage of galactose and lower levels of high-mannose glycans than were found on GP1. These results confirm and expand previous findings on partial characterisation of the Ebola virus transmembrane glycoprotein. They represent the first detailed data on carbohydrate structures of the Ebola virus sGP.

Figure 1

(a) Normal-phase separation of N-glycans present in GP1-HA from Ebola virus. (b) HPLC chromatogram after digestion with Arthrobacter ureafaciens sialidase (ABS) showing negligible sialic acid substitution, (c) after digestion with ABS and almond meal α-fucosidase (AMF) showing no fucose attached to the antennae, (d) ABS and bovine testis β-galactosidase (BTG) showing release of galactose residues, (e) ABS, BTG and β-N-acetylhexosaminidase (JBH) which reduces all complex glycans to core-fucosylated Man₃GlcNAc₂, and finally (f) ABS, BTG, JBH and bovine kidney α-fucosidase (BKF) showing the presence of core fucose. Numbers over the peaks in (a) refer to the structures in Table 1. Lines connecting the chromatograms show the movement of peaks after digestion. Symbols used for the structural formulae in this figure, other figures and Table 1: ■ = GlcNAc, ○ =mannose, ◇, = galactose, ⟐ = fucose. The angle of the lines connecting the symbols shows the linkage with full and broken lines specifying β- and α-linkages, respectively. The system is described in more detail in Harvey et al.

Figure 2

(a) Normal-phase separation of N-glycans present in sGP-HA from Ebola virus. Chromatograms (b–f) were produced by digestions with the same exoglycosidases as specified in the legend to Fig. 1. Lines connecting the chromatograms show the movement of peaks after digestion. Symbols used for the structural formulae are defined in the legend to Fig. 1.

Figure 3

MALDI-TOF spectra (from DHB) of the desialylated N-glycans released from (a) GP1-HA and (b) sGP-HA. Symbols used for the structural formulae are defined in the legend to Fig. 1. Spectra have been smoothed (Savitzky Golay 2 × 2) and processed with the MaxEnt 2 function of MassLynx to improve resolution. Ions around m/z 1200 in the lower spectrum are from contaminants. In addition, two very abundant ions from additional contaminants at m/z 1373 and 1712 in the lower spectrum have been removed for clarity. Ions are labelled with their monoisotopic masses.

Figure 4

Negative ion CID spectra of fucosylated biantennary glycans with (a) two, (b) one, and (c) no galactose residues on the antennae.

Figure 5

Negative ion CID spectra of fucosylated triantennary glycans with (a) three, (b) two, (c) one, and (d) no galactose residues on the antennae.

Figure 6

Negative ion CID spectra of high-mannose N-glycans from Ebola virus GP1-HA glycoprotein. Panels (a–e) show the spectra of Man₅GlcNAc₂ through Man₉GlcNAc₂. Structures of the isomers are shown. The broken lines connecting the spectra show the shifts in the fragment ions that define the isomer structures (see text).

Figure 7

(a) Normal-phase separation of O-glycans present in GP1-HA from Ebola virus. (b) HPLC chromatogram after digestion with Arthrobacter ureafaciens sialidase (ABS) showing sialic acid substitution, (c) after digestion with ABS and Streptococcus pneumoniae β-galactosidase (SPG, specific for β-(1→4)-linked galactose). The arrows show how the peaks move after digestion.