Wilcox W C, Long D, Sodora D L, Eisenberg R J, Cohen G H
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6003.
J Virol. 1988 Jun;62(6):1941-7. doi: 10.1128/JVI.62.6.1941-1947.1988.
Glycoprotein D (gD) is an envelope component of herpes simplex virus types 1 (gD-1) and 2 (gD-2). The gD-1 polypeptide contains seven cysteine residues among its 369 amino acids; six are located on the N-terminal or luminal portion of the glycoprotein, and a seventh is located in the transmembrane region. Previous studies used a panel of monoclonal antibodies (MAbs) to define gD epitopes as continuous or discontinuous. Purified gD, denatured by reduction and alkylation, loses discontinuous epitopes, whereas continuous epitopes are retained. The contribution of disulfide bonds to maintenance of discontinuous epitopes is, therefore, significant. In the present study, our objective was to determine the contribution of individual cysteine residues to folding of gD-1 into its native conformation. Site-directed oligonucleotide mutagenesis was used to create seven mutants, each with a serine residue replacing a cysteine. The mutated genes were cloned into a eucaryotic expression vector and transfected into COS-1 cells, and the proteins were separated by nondenaturing polyacrylamide gel electrophoresis, followed by immunoblotting. Replacement of cysteine 7 (residue 333) had only a minimal effect on the antigenic properties of gD-1. In contrast, replacement of any one of the other six cysteine residues resulted in either a major reduction or a complete loss of binding of those MAbs that recognize discontinuous epitopes, with no effect on the binding of MAbs which recognize continuous epitopes. These mutations also had profound effects on the extent of oligosaccharide processing of gD-1. This was determined by digestion of the expressed proteins with various endoglycosidases, followed by electrophoresis and Western blotting (immunoblotting) to observe any mobility changes. Three mutant gD proteins which did not express discontinuous epitopes contained only high-mannose-type oligosaccharides, suggesting that processing had not proceeded beyond the precursor stage. Two mutant forms of gD exhibited reduced binding of MAbs to discontinuous epitopes. A small proportion of the molecules which accumulated at 48 h posttransfection contained complex oligosaccharides. One mutant exhibited reduced binding of MAbs to discontinuous epitopes, but was present at 48 h posttransfection only in the precursor form. The cysteine 7 mutant was processed to the same extent as wild-type gD. We conclude that the first six cysteine residues are critical to the correct folding, antigenic structure, and processing of gD-1, and we speculate that they form three disulfide-bonded pairs.
糖蛋白D（gD）是单纯疱疹病毒1型（gD - 1）和2型（gD - 2）的包膜成分。gD - 1多肽在其369个氨基酸中含有7个半胱氨酸残基；其中6个位于糖蛋白的N端或腔内部分，第7个位于跨膜区域。先前的研究使用一组单克隆抗体（MAb）将gD表位定义为连续或不连续的。经还原和烷基化变性的纯化gD会失去不连续表位，而连续表位则得以保留。因此，二硫键对维持不连续表位具有重要作用。在本研究中，我们的目的是确定单个半胱氨酸残基对gD - 1折叠成其天然构象的贡献。使用定点寡核苷酸诱变创建了7个突变体，每个突变体都用丝氨酸残基取代一个半胱氨酸。将突变基因克隆到真核表达载体中并转染到COS - 1细胞中，然后通过非变性聚丙烯酰胺凝胶电泳分离蛋白质，接着进行免疫印迹。半胱氨酸7（第333位残基）的取代对gD - 1的抗原特性影响极小。相比之下，其他6个半胱氨酸残基中的任何一个被取代，都会导致那些识别不连续表位的单克隆抗体的结合大幅减少或完全丧失，而对识别连续表位的单克隆抗体的结合没有影响。这些突变对gD - 1的寡糖加工程度也有深远影响。这是通过用各种内切糖苷酶消化表达的蛋白质，然后进行电泳和Western印迹（免疫印迹）来观察迁移率变化来确定的。三种不表达不连续表位的突变gD蛋白仅含有高甘露糖型寡糖，这表明加工尚未超过前体阶段。两种gD突变形式对单克隆抗体与不连续表位的结合表现出降低。在转染后48小时积累的一小部分分子含有复合寡糖。一种突变体对单克隆抗体与不连续表位的结合表现出降低，但在转染后48小时仅以前体形式存在。半胱氨酸7突变体的加工程度与野生型gD相同。我们得出结论，前6个半胱氨酸残基对gD - 1的正确折叠、抗原结构和加工至关重要，并且我们推测它们形成了三对二硫键连接对。
