Structure-function analysis of the soluble glycoprotein, sGP, of Ebola virus.
In addition to the transmembrane protein, GP(1,2), the Ebola virus glycoprotein gene encodes the soluble glycoproteins sGP and Delta-peptide. Two more soluble proteins, GP(1) and GP(1,2DeltaTM), are generated from GP(1,2) as a result of disulfide-bond instability and proteolytic cleavage, respectively, and are shed from the surface of infected cells. The sGP glycoprotein is secreted as a disulfide-linked homodimer, but there have been conflicting reports on whether it is arranged in a parallel or antiparallel orientation. Off-line HPLC-MALDI-TOF MS (MS/MS) was used to identify the arrangement of all disulfide bonds and simultaneously determine site-specific information regarding N-glycosylation. Our data prove that sGP is a parallel homodimer that contains C53-C53' and C306-C306' disulfide bonds, and although there are six predicted N-linked carbohydrate sites, only five are consistently glycosylated. The disulfide bond arrangement was confirmed by using cysteine to glycine mutations at amino acid positions 53 and 306. The mutants had a reduced ability to rescue the barrier function of TNF-alpha-treated endothelial cells--a function previously reported for sGP. This indicates that these disulfide bonds are critical for the proposed anti-inflammatory function of sGP.
Falzarano
Darryl
D
Department of Medical Microbiology, University of Manitoba Winnipeg, Manitoba R3E 0W3, Canada.
Krokhin
Oleg
O
Wahl-Jensen
Victoria
V
Seebach
Jochen
J
Wolf
Kristin
K
Schnittler
Hans-Joachim
HJ
Feldmann
Heinz
H
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D029043:Ebolavirus / Q000737:chemistry*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus; D003545:Cysteine