Rux A H, Moore W T, Lambris J D, Abrams W R, Peng C, Friedman H M, Cohen G H, Eisenberg R J
Department of Microbiology, University of Pennsylvania, Philadelphia 19104, USA.
J Virol. 1996 Aug;70(8):5455-65. doi: 10.1128/JVI.70.8.5455-5465.1996.
A biochemical analysis of glycoprotein C (gC of herpes simplex virus was undertaken to further characterize the structure of the glycoprotein and to determine its disulfide bond arrangement. We used three recombinant forms of gC, gC1(457t), gC1(delta33-123t), and gC2(426t), each truncated prior to the transmembrane region. The proteins were expressed and secreted by using a baculovirus expression system and have been shown to bind to monoclonal antibodies which recognize discontinuous 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(delta33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(delta33-123t), 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-linked oligosaccharide sites on gC1(457t) are occupied by glycans of approximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acids [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosaccharides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t),the protein was cleaved with cyanogen bromide. SDS-PAGE analysis followed 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 127) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cysteine 4 (aa 347). Further proteolysis of the cyanogen bromide-generated fragment containing cysteine 5 through cysteine 8, combined with mass spectrometry and Edman degradation, showed that disulfide bonds link cysteine 5 (aa 386) to cysteine 8 (aa 442) and cysteine 6 (aa 390) to cysteine 7 (aa 419). A similar disulfide bond arrangement is postulated to exist in gC homologs from other herpesviruses.
对单纯疱疹病毒糖蛋白C（gC）进行了生化分析，以进一步表征该糖蛋白的结构并确定其二硫键排列。我们使用了三种gC的重组形式，gC1(457t)、gC1(δ33 - 123t)和gC2(426t)，每种在跨膜区域之前都被截短。这些蛋白质通过杆状病毒表达系统进行表达和分泌，并已显示能与识别不连续表位的单克隆抗体结合，并以剂量依赖方式与补体成分C3b结合。我们通过埃德曼降解法确认了每种成熟蛋白的N端残基，并确认了gC1(δ33 - 123t)中的内部缺失。通过用内切糖苷酶处理每种蛋白质，然后进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和质谱分析，确定了gC1(457t)、gC1(δ33 - 123t)和gC2(426t)的分子量和糖基化程度。数据表明，gC1(457t)上预测的8至9个N - 连接寡糖位点被约1000 Da的聚糖占据。此外，gC1(457t)上存在O - 连接寡糖，主要定位于该蛋白的N端区域（氨基酸[aa]33至123）。gC2(426t)含有N - 连接寡糖，但未检测到O - 连接寡糖。为了确定gC1(457t)的8个半胱氨酸的二硫键排列，该蛋白用溴化氰裂解。随后进行SDS - PAGE分析并结合埃德曼降解法，鉴定出三个不含通过二硫键连接的半胱氨酸片段。半胱氨酸的化学修饰与基质辅助激光解吸电离质谱联用，确定了半胱氨酸1（aa 127）和半胱氨酸2（aa 144）之间以及半胱氨酸3（aa 286）和半胱氨酸（aa 347）之间的二硫键。对含有半胱氨酸5至半胱氨酸8的溴化氰生成的片段进行进一步蛋白酶解，结合质谱和埃德曼降解法，表明二硫键将半胱氨酸5（aa 386）与半胱氨酸8（aa 442）以及半胱氨酸6（aa 390）与半胱氨酸7（aa 419）连接起来。推测其他疱疹病毒的gC同源物中存在类似的二硫键排列。
