ROLE OF INDIVIDUAL CYSTEINE RESIDUES IN THE PROCESSING AND ANTIGENICITY OF THE MEASLES-VIRUS HEMAGGLUTININ PROTEIN
ROLE OF INDIVIDUAL CYSTEINE RESIDUES IN THE PROCESSING AND ANTIGENICITY OF THE MEASLES-VIRUS HEMAGGLUTININ PROTEIN
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DOI：
10.1099/0022-1317-75-9-2173
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发表时间：
1994-09-01
影响因子：
3.8
通讯作者：
NORRBY, E
中科院分区：
文献类型：
--
作者：
HU, AH;NORRBY, E
血凝素（H）蛋白是麻疹病毒的主要包膜糖蛋白。该蛋白含有13个半胱氨酸残基，在其617个氨基酸中，全部位于其胞外域。在先前的研究中，定义了一组单克隆抗体（MAb）与连续和不连续表位反应的能力。结果表明，二硫键的缺乏损害了蛋白质与特异性针对不连续表位的单克隆抗体反应的能力。在本研究中，我们的目标是确定单个半胱氨酸残基的H蛋白折叠成其天然构象的贡献。定点寡核苷酸诱变用于产生13个突变体，每个突变体用丝氨酸替换半胱氨酸。通过使用牛痘病毒驱动的T7聚合酶系统在BHK-21细胞中直接表达突变基因。突变蛋白的抗原结构和细胞内加工特性的研究揭示了以下结果。(i)半胱氨酸残基139，154，188，386，570或606的替换对H蛋白的抗原结构和细胞内加工没有可检测的影响。然而，具有取代的半胱氨酸残基154的突变体显示出修饰的迁移特性。(ii)半胱氨酸残基381或494的改变对H蛋白的性质表现出中等的影响。这两个突变体表达不连续的表位，表明它们是部分折叠的，但它们没有寡聚化，没有到达内侧高尔基复合体，未能被转运到细胞表面。(iii)半胱氨酸残基287、300、394、579或583的取代导致识别不连续表位的MAb的结合完全丧失，对与连续表位反应的MAb的结合没有影响。没有观察到蛋白质的二聚体形式，并且在这些突变体中仅证明了高甘露糖寡糖，这表明修饰的蛋白质没有寡聚化并且保留在内质网中。总之，半胱氨酸残基287，300，381，394，494，579和583似乎在H分子的抗原结构和加工中发挥特别关键的作用，它们可能参与分子间或分子内的二硫键。
The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter- or intramolecular disulphide bonding.