Sato T A, Enami M, Kohama T
Department of Virus Disease and Vaccine Control, National Institute of Health, Tokyo, Japan.
J Virol. 1995 Jan;69(1):513-6. doi: 10.1128/JVI.69.1.513-516.1995.
The hemagglutinin (H) glycoprotein was isolated in a soluble form by digesting measles virus particles with an endoproteinase, Asp-N (from a Pseudomonas fragi mutant). Digestion of H with Asp-N brought about glycopeptides in three different forms, depending on the cleaving site: AHD, which has an M(r) of 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which formed a disulfide-linked homodimer with an M(r) of 132,000, and two monomeric digestion products, AHM-1 (with an M(r) of 64,000) and AHM-2 (with an M(r) of 58,000). The susceptibility of the H glycoprotein to the protease depended on the enzyme concentration. AHD was readily formed at a low concentration of Asp-N, while AHM-1 and AHM-2 required higher and even higher protease concentrations, respectively. All of the cleavage products reacted with monoclonal antibodies to various epitopes of the H protein; however, only AHD showed a significant hemagglutinin activity on African green monkey erythrocytes. The hemagglutinin activities of AHM-1 and AHM-2 were restored after a monoclonal antibody lacking the hemagglutination-inhibiting activity was added to the reaction mixture. AHDs purified by size-exclusion high-pressure liquid chromatography had two associating forms; one had an M(r) higher than and the other an M(r) as high as that of a tetramer. The former was associated noncovalently in addition to having two intermolecular disulfide bonds, and the latter was associated covalently with a single intermolecular disulfide bond and was also duplicated through a noncovalent association. In addition, both AHM-1 and AHM-2, having no intermolecular disulfide bond, were in a dimer form. These results suggest that AHM-1 and AHM-2 are monovalent in the hemagglutinin activity, while AHDs are divalent. Comparative analyses of the N termini of these soluble glycopeptides with the sequence of H suggested that the cysteine residue at position 139 was responsible for the intermolecular disulfide bonding between the monomeric H glycoproteins. The cysteine at position 154 was also suggested to participate in the forming of the intermolecular disulfide bond.
通过用一种内蛋白酶天冬氨酸蛋白酶N（来自脆弱假单胞菌突变体）消化麻疹病毒颗粒，以可溶形式分离出血凝素（H）糖蛋白。用天冬氨酸蛋白酶N消化H产生了三种不同形式的糖肽，这取决于切割位点：AHD，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定其相对分子质量（M(r)）为66,000，并且形成了相对分子质量为132,000的二硫键连接的同型二聚体，以及两种单体消化产物，AHM-1（相对分子质量为64,000）和AHM-2（相对分子质量为58,000）。H糖蛋白对蛋白酶的敏感性取决于酶浓度。在低浓度的天冬氨酸蛋白酶N下容易形成AHD，而AHM-1和AHM-2分别需要更高甚至更高的蛋白酶浓度。所有的切割产物都能与针对H蛋白各种表位的单克隆抗体发生反应；然而，只有AHD对非洲绿猴红细胞显示出显著的血凝活性。在反应混合物中加入缺乏血凝抑制活性的单克隆抗体后，AHM-1和AHM-2的血凝活性得以恢复。通过尺寸排阻高压液相色谱纯化的AHD有两种缔合形式；一种的相对分子质量高于四聚体，另一种的相对分子质量与四聚体一样高。前者除了有两个分子间二硫键外还通过非共价缔合，而后者通过单个分子间二硫键共价缔合，并且也通过非共价缔合重复。此外，没有分子间二硫键的AHM-1和AHM-2都呈二聚体形式。这些结果表明，AHM-1和AHM-2在血凝活性方面是单价的，而AHD是二价的。对这些可溶性糖肽的N端与H序列进行比较分析表明，第139位的半胱氨酸残基负责单体H糖蛋白之间的分子间二硫键结合。也有人认为第154位的半胱氨酸参与了分子间二硫键的形成。
