Cognet Isabelle, Guilhot Florence, Gabriac Mélanie, Chevalier Sylvie, Chouikh Younes, Herman-Bert Alexandra, Guay-Giroux Angélique, Corneau Sophie, Magistrelli Giovanni, Elson Greg C, Gascan Hugues, Gauchat Jean-François
Département de pharmacologie, Université de Montréal, C.P 6128, succursale Centre-ville, Montreal, QC, Canada H3C 3J7.
J Immunol Methods. 2005 Jun;301(1-2):53-65. doi: 10.1016/j.jim.2005.03.012. Epub 2005 Apr 26.
The recently identified IL-6 family member cardiotrophin-like cytokine (also named novel neurotrophin-1 or B cell stimulating factor-3) forms a secreted complex with cytokine-like factor-1 which binds and activates the tripartite ciliary neurotrophic factor receptor. The striking differences between the phenotype of mice in which either the ciliary neurotrophic factor or its receptor are inactivated suggest that the cardiotrophin-like cytokine/cytokine-like factor-1 complex could be the developmentally important ciliary neurotrophic factor receptor ligand. Cardiotrophin-like cytokine is also produced in the immune system and has been reported to activate B cells in vivo and in vitro. B cells do not express the ciliary neurotrophic factor receptor suggesting the existence of an alternative receptor. We produced the cardiotrophin-like cytokine/cytokine-like factor-1 complex tagged with a Bir A biotin ligase AviTag peptide substrate. This cytokine could be efficiently biotinylated in vitro with Bir A. It was subsequently validated as a sensitive tool for ciliary neurotrophic factor receptor detection by flow cytometry and for magnetic-activated cell sorting. It was also shown to allow the detection of a specific receptor by activated B cells. Whereas binding to cells expressing the ciliary neurotrophic factor receptor could be prevented by competition with ciliary neurotrophic factor, binding to B cells was not. The biotinylated cardiotrophin-like cytokine/cytokine-like factor-1 complex therefore represents a new reagent to study ciliary neurotrophic factor and cardiotrophin-like cytokine receptor expression and for the identification of the putative cardiotrophin-like cytokine B cell receptor. It further validates the use of biotin ligase catalysed biotinylation for the detection of cytokine receptors.
最近鉴定出的白细胞介素-6家族成员心肌营养素样细胞因子（也称为新型神经营养因子-1或B细胞刺激因子-3）与细胞因子样因子-1形成一种分泌复合物，该复合物可结合并激活三联睫状神经营养因子受体。睫状神经营养因子或其受体失活的小鼠的表型存在显著差异，这表明心肌营养素样细胞因子/细胞因子样因子-1复合物可能是在发育过程中起重要作用的睫状神经营养因子受体配体。心肌营养素样细胞因子也在免疫系统中产生，并且据报道在体内和体外均可激活B细胞。B细胞不表达睫状神经营养因子受体，这表明存在替代受体。我们制备了带有Bir A生物素连接酶AviTag肽底物标签的心肌营养素样细胞因子/细胞因子样因子-1复合物。这种细胞因子在体外可以被Bir A有效地生物素化。随后，它被验证为通过流式细胞术检测睫状神经营养因子受体以及用于磁珠激活细胞分选的灵敏工具。它还被证明可以检测激活的B细胞的特异性受体。与表达睫状神经营养因子受体细胞的结合可通过与睫状神经营养因子竞争来阻止，而与B细胞的结合则不然。因此，生物素化的心肌营养素样细胞因子/细胞因子样因子-1复合物是一种研究睫状神经营养因子和心肌营养素样细胞因子受体表达以及鉴定推定的心肌营养素样细胞因子B细胞受体的新试剂。它进一步验证了生物素连接酶催化的生物素化用于检测细胞因子受体的用途。
