Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.
Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.
复制标题
仙台病毒血凝素神经氨酸酶蛋白的生物活性可溶形式的分离。
DOI：
10.1128/jvi.62.12.4653-4660.1988
复制
发表时间：
1988
影响因子：
5.4
通讯作者：
Portner,A
中科院分区：
文献类型：
--
作者：
Thompson,SD;Laver,WG;Murti,KG;Portner,A
作为建立仙台病毒血凝素-神经氨酸酶（HN）三维结构的第一步，我们已经分离并表征了该分子的潜在结晶形式。HN是一种表面糖蛋白，其序列预测了一种在氨基末端附近具有不带电荷的疏水区域的蛋白质，该区域负责锚定在病毒包膜中。为了避免会妨碍结晶的玫瑰花结形成（聚集），通过蛋白水解消化从无膜形式的HN中除去该疏水尾部。该消化产生分子量比天然HN小约10，000的单一产物。切割的HN（C-HN）的N-末端氨基酸序列分析表明在氨基酸残基131处的单个切割位点，产生由HN的羧基末端444个氨基酸组成的产物。功能分析表明，C-HN保留了完整的神经氨酸酶活性，并能够结合红细胞，表明N-末端131个残基是不必要的这些生物活性。此外，这种切割产物保留了完整HN的抗原结构，因为单克隆抗体在酶联免疫吸附测定和Western（免疫）印迹分析中仍然与C-HN结合。通过电子显微镜观察，完整HN的二聚体和四聚体形式形成棒状，而C-HN保持低聚体结构但不再聚集。此外，电子显微照片显示C-HN四聚体在大小和大体结构特征上与流感病毒神经氨酸酶惊人相似。
As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features.