Mirza A M, Sheehan J P, Hardy L W, Glickman R L, Iorio R M
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
J Biol Chem. 1993 Oct 5;268(28):21425-31.
The hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses is anchored in the virion membrane near its amino terminus, protruding from the virion surface to mediate attachment to cellular receptors. Solubilization of HN spikes can be achieved by treatment of virions with detergent and high salt concentrations. When the solubilized HN protein from the Australia-Victoria (AV) isolate of the virus is incubated at 37 degrees C, a chymotrypsin-sensitive site between residues 112 and 113 is exposed. A chymotrypsin-cleaved soluble form of the protein, named CT-HN, has been prepared using this approach. It is membrane anchor-less, due to removal of a 14-kDa fragment from the NH2 terminus of HN. It retains all potential glycosylation sites and cysteines present in the ectodomain of the native protein. It migrates in nonreducing gels and sediments in sucrose gradients at the rate expected for homodimeric HN. The latter is also consistent with our demonstration by site-directed mutagenesis that cysteine residues at positions 6 and 123, respectively, mediate disulfide-linked homotetramer and homodimer formation. CT-HN retains almost total antigenicity, suggesting that it is conformationally very similar to the intact molecule, as well as receptor recognition function and, at low pH, neuraminidase activity. It should prove to be a useful tool for further studies of the structure and function of this important viral glycoprotein.
副粘病毒的血凝素神经氨酸酶（HN）糖蛋白在病毒粒子膜中靠近其氨基末端处锚定，从病毒粒子表面突出以介导与细胞受体的附着。通过用去污剂和高盐浓度处理病毒粒子可实现HN刺突的溶解。当将来自该病毒澳大利亚维多利亚（AV）分离株的溶解的HN蛋白在37℃孵育时，112和113位残基之间的一个糜蛋白酶敏感位点暴露出来。使用这种方法制备了一种名为CT-HN的蛋白的糜蛋白酶切割可溶性形式。由于从HN的NH2末端去除了一个14 kDa的片段，它没有膜锚定结构。它保留了天然蛋白胞外域中存在的所有潜在糖基化位点和半胱氨酸。它在非还原凝胶中迁移，并以同源二聚体HN预期的速率在蔗糖梯度中沉降。后者也与我们通过定点诱变证明的6位和123位的半胱氨酸残基分别介导二硫键连接的同源四聚体和同源二聚体形成一致。CT-HN几乎保留了全部抗原性，表明其构象与完整分子非常相似，同时还保留了受体识别功能以及在低pH下的神经氨酸酶活性。它应该被证明是进一步研究这种重要病毒糖蛋白结构和功能的有用工具。
