Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein
Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein
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DOI：
10.1128/jvi.78.19.10328-10335.2004
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发表时间：
2004-10-01
影响因子：
5.4
通讯作者：
Han, JH
中科院分区：
文献类型：
--
作者：
Song, HC;Seo, MY;Han, JH
我们已经表达了严重急性呼吸综合征冠状病毒（SARS-CoV）刺突蛋白的cDNA转染的哺乳动物细胞的特点。全长刺突蛋白（S）是新合成的一种内切糖苷酶H（endo H）敏感糖蛋白（gp 170），在高尔基体中进一步修饰为endo H抗性糖蛋白（gp 180）。没有观察到S的实质性蛋白水解裂解，表明S没有被加工成头部（S1）和茎部（S2）结构域，如在某些其他冠状病毒中观察到的那样。虽然表达的全长S糖蛋白是专门的细胞相关的，通过排除C-末端跨膜和胞质尾结构域的S截短导致内质网定位的糖蛋白（gp 160）以及高尔基体特异性形式（gp 170），最终分泌到细胞培养基中的表达。化学交联、热变性和大小分级分析表明，SARS-CoV的全长S糖蛋白形成了一个类似于500 kDa的高级结构，这与它是一个S同源三聚体相一致。在纯化的病毒体中也观察到后者。C-末端截短的S蛋白的细胞内形式（但不是分泌形式）也形成三聚体，但效率比全长S低得多。在天然条件下用肽N-糖苷酶-F对全长同源三聚体进行去糖基化，消除了对纯化病毒体的病毒中和抗血清对蛋白质的识别，表明碳水化合物在S蛋白正确折叠中的重要性。这些数据应有助于设计重组疫苗抗原，以防止这种新出现的病原体的传播。
We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that, S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of similar to500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by,virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.