Labeling of the cytoplasmic domain of the influenza virus hemagglutinin with fluorescein reveals sites of interaction with membrane lipid bilayers.
Labeling of the cytoplasmic domain of the influenza virus hemagglutinin with fluorescein reveals sites of interaction with membrane lipid bilayers.
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用荧光素标记流感病毒血凝素的细胞质结构域揭示了与膜脂双层相互作用的位点。
DOI：
10.1021/bi00348a043
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Parce,JW
中科院分区：
文献类型：
--
作者：
Lyles,DS;McKinnon,KP;Parce,JW
用荧光素标记流感病毒的血凝素（HA）糖蛋白的胞浆区。在外部域的反应性氨基被封闭的修饰的完整的病毒与膜不可渗透的试剂羟乙硫酰乙酰亚胺酯。然后用辛基葡糖苷溶解HA，并将胞质结构域中的单个赖氨酸与异硫氰酸荧光素反应。该方案导致1.3 mol荧光素/mol HA的掺入。使用在HA的胞质结构域中缺乏赖氨酸的病毒株，确定0.47 mol荧光素/mol HA位于另外的位点。在这两个网站上的荧光基团存在于一个环境中的极性降低所示的激发和发射最大值的位移和位移的pKa的荧光素基团。荧光偏振和荧光素基团的pKa值更大时，HA被纳入脂质体比在洗涤剂溶液中。这些数据表明，荧光素基团直接与脂质双层相互作用，可能在磷脂头基区域。标记的HA的荧光特性不响应于脂质双层中的凝胶到液晶相变。这些结果表明，细胞质结构域和锚定蛋白质的脂质双层的疏水序列之间的边界位于头基区的双层。流感病毒的膜状包膜含有两种表面糖蛋白[广泛综述，如参见Lamb（1983）]。血凝素（HA）1是主要的病毒粒子表面蛋白。它负责病毒附着到宿主细胞上含有唾液酸的受体上，并在病毒穿透期间负责病毒包膜与内吞囊泡膜的融合。神经氨酸酶（NA）糖蛋白以较少量存在于病毒包膜中，并负责从受体释放病毒。两种糖蛋白都在内质网中合成，并通过与宿主质膜糖蛋白相同的途径转运至宿主质膜。病毒包膜是通过从宿主细胞膜上出芽而获得的。在出芽过程中，内部病毒成分与膜的细胞质表面结合，并与病毒糖蛋白相互作用，形成一个专门的膜区域，宿主蛋白在很大程度上被排除在外。病毒表面糖蛋白是病毒包膜和宿主细胞膜上的主要抗原种类，抗病毒抗体可直接作用于其上。这些糖蛋白的周期性遗传变异产生了新的病毒株，这些病毒株在抗原性上是不同的，是流感反复流行的原因。HA糖蛋白是相同亚基的三聚体。每个亚基由两个二硫键连接的肽链组成，命名为HA 1（Mr约50000）和HA 2（Mr ap-proximate 25000），它们是通过共同前体HA 0的蛋白水解而衍生的。HA由美国国立卫生研究院（AI-15892和AI-20778）和美国癌症协会（IM-307）的研究赠款支持。信件应寄给作者。*微生物学和免疫学系。现住址：北卡罗来纳州达勒姆，杜克大学医学院微生物学系，邮编：27710。11生物化学
The hemagglutinin (HA) glycoprotein of influenza virus was labeled in its cytoplasmic domain with fluorescein. Reactive amino groups in the external domain were blocked by modification of the intact virus with the membrane-impermeable reagent isethionyl acetimidate. The HA was then solubilizedwith the detergent octyl glucoside, and the single lysine in the cytoplasmic domain was reacted with fluorescein isothiocyanate. This protocol resulted in the incorporation of 1.3 mol of fluorescein/mol of HA. Using a virus strain lacking lysine in the cytoplasmic domain ofHA, it was determined that 0.47 mol of fluor-escein/mol of HA was located at an additional site (s). The fluoresceingroups at both sites exist in an environment of reduced polarity as shown by a shift in excitation and emission maxima and a shift in the pKa of the fluorescein groups. The fluorescence polarization and the pKa of the fluorescein groups were greater when the HA was incorporated into liposomes than when in detergent solution. These dataindicate that the fluorescein groups interactdirectly with the lipid bilayer, probably in the phospholipid head-group region. The fluorescence properties of the labeled HA were not responsive to the gel to liquid-crystal phase transition in the lipid bilayer. These results indicate that the boundary between the cytoplasmic domain and the hydrophobic sequence that anchors the protein to the lipid bilayer is located in the head-group region of the bilayer. e membranelike envelope of influenza viruses contains two surface glycoproteins [reviewed extensively, eg, see Lamb (1983)]. The hemagglutinin (HA) 1 is the major virion surface protein. It is responsible for virus attachment to sialic acid containing receptors on host cells and for fusion of the virus envelope with the membrane of endocytic vesicles during virus penetration. The neuraminidase (NA) glycoprotein is present in lesser amounts in the virus envelope and is responsible for release of the virus from receptors. Both glycoproteins are synthesized in the endoplasmic reticulum and transported to the host plasma membrane by the same pathway followed by host plasma membrane glycoproteins. The virus envelope is acquired by budding from the host membrane. During the budding process, the internal viral components associate with the cytoplasmic surface of the membrane and interact with the viral glycoproteins to form a specialized region of the membrane from which host proteins are largely excluded. The surface glycoproteins are the major antigenic species on the virus envelope and host plasma membrane to which antiviral antibody is directed. Periodic genetic variation in these gly-coproteins gives rise to new virus strains that are antigenically distinct and are responsible for recurring epidemics of influ-enza. The HA glycoprotein is a trimer of identical subunits. Each subunit consists of two disulfide-linked peptidechains designated HA](Mr approximately 50000) and HA2 (Mr ap-proximately 25000) derived by proteolysis of a common precursor, HA0. The HA is anchored to the envelope phos-t Supported by research grants from the National Institutes of Health (Al-15892 and AI-20778) and the American Cancer Society (IM-307).* Correspondence should be addressed to this author.* Department of Microbiology and Immunology. s Present address: Department of Microbiology, Duke University School of Medicine, Durham, NC 27710. 11 Department of Biochemistry.