Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum apical membrane antigen 1
Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum apical membrane antigen 1
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DOI：
10.1128/iai.72.5.2947-2955.2004
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发表时间：
2004-05-01
影响因子：
3.1
通讯作者：
de Vries, E
中科院分区：
文献类型：
--
作者：
Gaffar, FR;Yatsuda, AP;de Vries, E
顶膜抗原1（AMA-1）是一种分泌于疟原虫裂殖子和弓形虫速殖子表面的微线蛋白，在宿主细胞侵袭中起重要作用。在这里，我们描述了克隆和表征的Bababelovis AMA-1同源指定BbAMA-1。BbAMA-1与恶性疟原虫AMA-1的总体相似性水平较低（18%），但AMA-1的典型特征，如C末端附近的跨膜结构域、预测的具有保守序列特性的短胞质C末端序列和含有14个保守半胱氨酸残基的胞外结构域，这些保守半胱氨酸残基参与二硫键形成。兔多克隆抗血清提出了对三个合成肽来自N-末端区域和结构域II和III的推定的胞外结构域，并显示出识别特异性重组BbAMA-1在大肠杆菌中表达。免疫荧光显微镜显示，有标记的顶端一半的裂殖子与这些抗血清。预孵育的自由裂殖子与所有三种抗血清的入侵红细胞的效率降低了65%的最大值。针对N-末端肽的抗血清在Western印迹上检测到82-kDa的蛋白质，在体外侵袭后收获的上清液中检测到69-kDa的蛋白质，这表明蛋白水解加工和分泌发生在侵袭期间或侵袭后不久。二维蛋白质印迹和代谢标记的组合允许直接鉴定与BbAMA-1肽抗血清反应的斑点以及这些斑点的非常低的银染色强度，表明巴氏巴氏裂殖子中存在非常低水平的BbAMA-1。
Apical membrane antigen 1 (AMA-1) is a micronemal protein secreted to the surface of merozoites of Plasmodium species and Toxoplasma gondii tachyzoites in order to fulfill an essential but noncharacterized function in host cell invasion. Here we describe cloning and characterization of a Babesia bovis AMA-1 homologue designated BbAMA-1. The overall level of similarity of BbAMA-1 to P. falciparum AMA-1 was low (18%), but characteristic features like a transmembrane domain near the C terminus, a predicted short cytoplasmic C-terminal sequence with conserved sequence properties, and an extracellular domain containing 14 conserved cysteine residues putatively involved in disulfide bridge formation are typical of AMA-1. Rabbit polyclonal antisera were raised against three synthetic peptides derived from the N-terminal region and domains II and III of the putative extracellular domain and were shown to recognize specifically recombinant BbAMA-1 expressed in Escherichia coli. Immunofluorescence microscopy showed that there was labeling of the apical half of merozoites with these antisera. Preincubation of free merozoites with all three antisera reduced the efficiency of invasion of erythrocytes by a maximum of 65%. Antisera raised against the N-terminal peptide detected a 82-kDa protein on Western blots and a 69-kDa protein in the supernatant that was harvested after in vitro invasion, suggesting that proteolytic processing and secretion take place during or shortly after invasion. A combination of two-dimensional Western blotting and metabolic labeling allowing direct identification of spots reacting with the BbAMA-1 peptide antisera together with the very low silver staining intensity of these spots indicated that very low levels of BbAMA-1 are present in Babesia merozoites.