A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins
A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins
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DOI：
10.1046/j.1365-3024.2001.00366.x
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发表时间：
2001-03-01
影响因子：
2.2
通讯作者：
Pritchard, DI
中科院分区：
文献类型：
--
作者：
Kasper, G;Brown, A;Pritchard, DI
钙网蛋白最近被鉴定为美洲钩虫变应原，意味着与免疫系统细胞的分泌和接触，或宿主组织中显著的蠕虫磨损。由于人类钙网织蛋白已被证明与补体成分C1q的溶血活性结合并中和，并可能参与整合素介导的血小板内信号事件，因此有兴趣确定来自成功的人类线虫寄生虫的钙网织蛋白是否具有已知的免疫调节和抗止血特性，能够干扰补体的激活并与血小板和其他白细胞中与细胞信号相关的整合素域相互作用。我们现在可以报告，重组的钙网蛋白没有表现出显著的钙结合能力，这是钙网蛋白的一个特征，可能表明在原核生物中表达后不适当的折叠。然而，重组钙网状蛋白保留了足够的分子结构来与人C1q结合并抑制其溶血能力。此外，重组钙网蛋白在表面等离子共振分析(SPR)中与整合素αIIb和α5(，)的胞质信号域相对应的多肽以不依赖钙的方式发生反应。用SPR鉴定钩虫钙网织蛋白多克隆抗体的特异性，再用SPR鉴定天然分子表达的阶段特异性(与逆转录聚合酶链式反应比较)，以指示其明显分泌，并用亲和层析从虫体提取物中纯化天然钙网织蛋白。这一进展将允许对天然钙网蛋白重复上述功能测试，以确定其在宿主-寄生虫关系中的作用。
Calreticulin was recently identified as a hookworm Necator americanus allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host. As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin-mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes. We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote. Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q. Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins alpha IIb and alpha5(,) in a calcium independent manner. SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase-polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography. This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host-parasite relationship.