Structure-function analysis of the soluble glycoprotein, sGP, of ebola virus

Abstract

In addition to the transmembrane protein, GP1,2, the Ebola virus glycoprotein gene encodes the soluble glycoproteins sGP and Δ-peptide. Two more soluble proteins, GP1 and GP1,2ΔTM, are generated from GP1,2 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-α-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. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.