Akpek Esen K, Liu Sammy H, Thompson Robert, Gottsch John D
Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.
Invest Ophthalmol Vis Sci. 2002 Aug;43(8):2677-84.
Calgranulin C (CaGC) is a protein released by activated neutrophils and involved in host defense against filarial infections. This study involved the identification of binding protein(s) of the helminth Brugia malayi to CaGC and the ability of binding complexes to induce keratitis.
Parasitic extracts prepared from B. malayi microfilariae and adult worms were incubated with recombinant CaGC protein. Parasite binding protein-CaGC complex was isolated by affinity chromatography. A B. malayi microfilariae cDNA library was immunoscreened with antisera from rats immunized with the isolated parasitic CaGC-binding protein. All positive clones contained paramyosin sequences. Paramyosin was thus considered the major CaGC-binding protein in the parasite. To delineate the binding of CaGC to native and recombinant paramyosin, 125I-CaGC was used as a binding tracer in SDS-PAGE analysis to identify a CaGC-binding complex. To determine whether the complex of CaGC and its binding protein could induce keratitis mimicking the onchocercal human corneal disease, BALB/c mice preimmunized with the binding complex were challenged with intracorneal binding complex or live Brugia microfilariae. In addition, splenocytes harvested from the same animals were assessed for their ability to elicit cellular immune responses to the binding complex by [3H]thymidine assay.
In vitro binding of CaGC to paramyosin was confirmed using recombinant paramyosin and 125I-CaGC. Test animals showed development of severe keratitis that mimicked, clinically and histopathologically, the human onchocercal corneal disease, demonstrating the antigenic specificity of the paramyosin-CaGG-binding complex.
Paramyosin is identified as a CaGC-binding protein in B. malayi.
钙粒蛋白C（CaGC）是一种由活化的中性粒细胞释放的蛋白质，参与宿主对丝虫感染的防御。本研究旨在鉴定马来布鲁线虫与CaGC的结合蛋白以及结合复合物诱导角膜炎的能力。
将从马来布鲁线虫微丝蚴和成虫制备的寄生虫提取物与重组CaGC蛋白孵育。通过亲和层析分离寄生虫结合蛋白-CaGC复合物。用来自用分离的寄生虫CaGC结合蛋白免疫的大鼠的抗血清对马来布鲁线虫微丝蚴cDNA文库进行免疫筛选。所有阳性克隆均包含副肌球蛋白序列。因此，副肌球蛋白被认为是寄生虫中主要的CaGC结合蛋白。为了描述CaGC与天然和重组副肌球蛋白的结合，在SDS-PAGE分析中使用125I-CaGC作为结合示踪剂来鉴定CaGC结合复合物。为了确定CaGC及其结合蛋白的复合物是否能诱导模拟盘尾丝虫性人类角膜疾病的角膜炎，用结合复合物预免疫的BALB/c小鼠用角膜内结合复合物或活的马来布鲁线虫微丝蚴进行攻击。此外，通过[3H]胸苷测定评估从同一动物收获的脾细胞对结合复合物引发细胞免疫反应的能力。
使用重组副肌球蛋白和125I-CaGC证实了CaGC在体外与副肌球蛋白的结合。试验动物出现了严重的角膜炎，在临床和组织病理学上模拟了人类盘尾丝虫性角膜疾病，证明了副肌球蛋白-CaGG结合复合物的抗原特异性。
副肌球蛋白被鉴定为马来布鲁线虫中的一种CaGC结合蛋白。
