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Published on: December 24, 2016
Cardiotrophin-like cytokine labelling using Bir A biotin ligase: a sensitive tool to study receptor expression by
Isabelle Cognet1, Florence Guilhot, Mélanie Gabriac
1Département de pharmacologie, Université de Montréal, C.P 6128, succursale Centre-ville, Montreal, QC, Canada H3C 3J7.
Insights
A novel biotinylated complex of cardiotrophin-like cytokine and cytokine-like factor-1 aids in detecting cytokine receptors on B cells and other cells, advancing research on immune cell signaling.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Cardiotrophin-like cytokine (CLC), an IL-6 family member, forms a complex with cytokine-like factor-1 (CLF-1).
- This CLC/CLF-1 complex binds the ciliary neurotrophic factor (CNTF) receptor, but B cells, which respond to CLC, lack this receptor, suggesting an alternative receptor.
- Differences in mouse phenotypes upon CNTF or its receptor inactivation suggest the CLC/CLF-1 complex is a key CNTF receptor ligand.
Purpose of the Study:
- To develop a tool for detecting cytokine receptors, particularly on B cells.
- To investigate the interaction of the CLC/CLF-1 complex with its receptors.
- To validate biotin ligase-catalyzed biotinylation for cytokine receptor detection.
Main Methods:
- Produced a CLC/CLF-1 complex tagged with a Bir A biotin ligase AviTag.
- Biotinylated the tagged complex in vitro using Bir A.
- Validated the biotinylated complex for flow cytometry, magnetic-activated cell sorting, and receptor binding assays.
Main Results:
- The biotinylated CLC/CLF-1 complex efficiently detected CNTF receptor and a specific receptor on B cells.
- Binding to CNTF receptor-expressing cells was inhibited by CNTF, but binding to B cells was not.
- This demonstrates the utility of biotinylated CLC/CLF-1 for studying cytokine receptor expression and identifying the CLC B cell receptor.
Conclusions:
- The biotinylated CLC/CLF-1 complex is a valuable reagent for studying CNTF and CLC receptor expression.
- It facilitates the identification of the putative CLC B cell receptor.
- Biotin ligase-catalyzed biotinylation is validated as a method for detecting cytokine receptors.
Abstract:
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.
