Identification of N-glycans from Ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry.
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.
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK.
Gayle
G
Ritchie
David J
DJ
Harvey
Ute
U
Stroeher
Friederike
F
Feldmann
Heinz
H
Feldmann
Victoria
V
Wahl-Jensen
Louise
L
Royle
Raymond A
RA
Dwek
Pauline M
PM
Rudd
D002460:Cell Line; D029043:Ebolavirus / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000737:chemistry; D005690:Galactose / Q000737:chemistry; D006388:Hemagglutinins / Q000737:chemistry; D006801:Humans; D008358:Mannose / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000378:metabolism
D006388:Hemagglutinins; D011134:Polysaccharides; D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus; D005643:Fucose; D008358:Mannose; D005690:Galactose