Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo
Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo
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DOI：
10.1074/jbc.m111.300673
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发表时间：
2011-11-25
影响因子：
4.8
通讯作者：
Hartshorn, Kevan L.
中科院分区：
文献类型：
--
作者：
Crouch, Erika;Nikolaidis, Nikolaos;Hartshorn, Kevan L.
表面活性蛋白D（SP-D）对甲型流感病毒（LAY）的识别是通过SP-D碳水化合物识别结构域（CRD）与包膜糖蛋白上显示的聚糖之间的相互作用介导的。尽管天然人SP-D显示出有效的抗病毒和聚集活性，但三聚体重组颈+CRD（NCRD）显示出很少或没有影响IAV感染的能力。突变的三聚体NCRD，D325 A/R343 V，表现出显着的血凝抑制和病毒中和，与中性粒细胞的病毒聚集和聚集依赖性的病毒摄取。通过表面等离子体共振，D325 A/R343 V表现出与Phil 82血凝素三聚体（HA）的葡萄糖敏感性结合。相比之下，与来自HA头部上缺乏聚糖的另一种病毒（PR 8）的HA三聚体的结合非常低。质谱法证明在Phi 182 HA上已知有助于LAY结合的位置存在高甘露糖聚糖。分子建模预测HA聚糖和D325 A/R343 V之间桥接相互作用的能力增强。最后，三聚体D325 A/R343 V NCRD在使用具有更重糖基化HA的重组A/WSN/33病毒的IAV感染鼠模型中降低发病率并增加病毒清除。合并的数据支持一种模型，其中截短的突变体SP-D与LAY HA聚糖的结合改变促进病毒聚集，导致体外和体内显著的病毒中和。这些研究表明，潜在的效用的同源建模和蛋白质结构分析工程有效的聚集蛋白抗病毒药物作为体内治疗。
The recognition of influenza A virus (LAY) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck + CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phi182 HA at positions known to contribute to LAY binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to LAY HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.