A novel immunoprecipitation strategy identifies a unique functional mimic of the glial cell line-derived neurotrophic factor family ligands in the pathogen Trypanosoma cruzi.
A novel immunoprecipitation strategy identifies a unique functional mimic of the glial cell line-derived neurotrophic factor family ligands in the pathogen Trypanosoma cruzi.
复制标题
一种新的免疫沉淀策略鉴定了病原体克氏锥虫中神经胶质细胞系衍生的神经营养因子家族配体的独特功能模拟物。
DOI：
10.1128/iai.00411-08
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发表时间：
2008
影响因子：
3.1
通讯作者：
PereiraPerrin,Mercio
中科院分区：
文献类型：
--
作者：
Lu,Bo;PereiraPerrin,Mercio
The journey of the Chagas' disease parasiteTrypanosoma cruziin the human body usually starts in the skin after an insect bite, when trypomastigotes get through the extracellular matrix to bind specific surface receptors in the epidermis and dermis to enter cells, where they differentiate and replicate. As the infection spreads to the heart, nervous system, and other parts of the body via the circulatory system, the parasite must also cope with additional receptors in the immune system and vascular endothelium. The molecular underpinnings that govern host cell receptor recognition byT. cruzicounterreceptors remain largely unknown. Here, we describe an immunoprecipitation strategy designed to concurrently identify host receptors and complementing parasite counterreceptors. Extracellular domains of growth factor receptors fused to human immunoglobulin G (IgG) Fc were incubated with parasite lysates, immunoprecipitated on protein G-Sepharose, and eluted with Laemmli sample buffer. PossibleT. cruzicounterreceptors pulled down by the receptor-Fc bait were visualized on immunoblots probed with multispecific high-affinity IgG from chronic chagasic sera and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels stained with silver or Coomassie blue. In screening receptors important for nervous system repair, this parasite counterreceptor immunoprecipitation (PcIP) assay identified 7 to 11 polypeptides (molecular masses, 14 kDa to 55 kDa) that bound to the coreceptors of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) GFRα-1, -2, and -3. Binding was specific because theT. cruzimimic of host GFLs, named TGFL, did not react with GFL coreceptor tyrosine kinase RET and with other neurotrophic receptors. The polypeptides were located on the parasite outer membrane and bound noncovalently to each other. TGFL eluted from the GFL receptor/protein G affinity column with 0.5 M NaCl, pH 7.5, and potently promoted neurite outgrowth and cell survival in a GFL-sensitive mouse pheochromocytoma cell line. Given that GFLs are neuron survival factors crucial for development and maintenance of central and peripheral nervous systems, it may be thatT. cruzimimicry of host GFLs helps in mutually beneficial host repair of infected and damaged nervous tissue. As there are >30 growth factor receptor-Fc chimeras commercially available, this PcIP assay can be readily adapted to identify receptors/counterreceptors in otherT. cruziinvasion sites and in other infections such as Lyme disease, amebiasis, and schistosomiasis.
