DISULFIDE BOND STRUCTURE DETERMINATION AND BIOCHEMICAL-ANALYSIS OF GLYCOPROTEIN-C FROM HERPES-SIMPLEX-VIRUS

A biochemical analysis of glycoprotein C (gC) of herpes simplex virus was undertaken to further characterize the structure of the glycoprote in and to determine its disulfide bond arrangement. We used three reco mbinant forms of gC, gC1(457t), gC1(Delta 33-123t), and gC2(426t), eac h truncated prior to the transmembrane region. The proteins were expre ssed and secreted by using a baculovirus expression system and have be en shown to bind to monoclonal antibodies which recognize discontinuou s epitopes and to complement component C3b in a dose-dependent manner. We confirmed the N-terminal residues of each mature protein by Edman degradation and confirmed the internal deletion in gC1(Delta 33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(D elta 33-123t), and gC2(426t) were determined by treating each protein with endoglycosidases and then subjecting it to sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. The data indicate that eight to nine of the predicted N-link ed oligosaccharide sites on gC1(457t) are occupied by glycans of appro ximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acid s [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosac charides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t), t he protein was cleaved with cyanogen bromide. SDS-PAGE analysis follow ed by Edman degradation identified three cysteine-containing fragments which are not connected by disulfide linkages. Chemical modification of cysteines combined with matrix-assisted laser desorption ionization mass spectrometry identified disulfide bonds between cysteine 1 (aa 1 27) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cystei ne 4 (aa 347). Further proteolysis of the cyanogen bromide-generated f ragment containing cysteine 5 through cysteine 8, combined with mass s pectrometry and Edman degradation, showed that disulfide bonds link cy steine 5 (aa 386) to cysteine 8 (aa 442) and cysteine 6 (aa 390) to cy steine 7 (aa 419). A similar disulfide bond arrangement is postulated to exist in gC homologs from other herpesviruses.