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Construction and characterization of ciliary neurotrophic factor (CNTF) antagonists: microenvironmental difference in
M Inoue1, H Karita, C Nakayama
1Sumitomo Pharmaceuticals Research Center, Konohana-ku, Osaka, Japan.
Abstract:
Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three-dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.
Insights
Engineered ciliary neurotrophic factor (CNTF) mutants show antagonistic properties, inhibiting neuronal survival. Specific mutations reveal differences in chicken versus rat receptor binding, impacting therapeutic potential.
Area of Science:
- Neuroscience
- Molecular Biology
- Protein Engineering
Background:
- Ciliary neurotrophic factor (CNTF) is crucial for neuronal survival.
- Understanding CNTF-receptor interactions is key for developing targeted therapies.
- Previous studies have not fully elucidated the role of specific residues in CNTF's activity.
Purpose of the Study:
- To construct and characterize antagonistic mutants of CNTF.
- To investigate the differential receptor recognition between chicken and rat neurons.
- To identify key residues and interactions involved in CNTF's biological activity.
Main Methods:
- Site-directed mutagenesis was used to create K155A, K155W, F152S/K155A, and F152D/K155A CNTF mutants.
- Cell survival assays were performed using chicken and rat dorsal root ganglion (DRG) neurons.
- Structural analysis of CNTF was used to infer binding interactions.
Main Results:
- K155A and K155W mutants lost chicken DRG neuron survival activity and inhibited wild-type CNTF.
- These mutants retained slight agonistic activity on rat DRG neurons, suggesting species-specific receptor recognition.
- Combined mutations (F152S/K155A, F152D/K155A) rendered CNTF antagonistic to both chicken and rat DRG neurons.
- Structural analysis indicated F152 and K155 are critical for receptor binding via hydrophobic and electrostatic interactions, respectively.
Conclusions:
- The D1 cap region, including residues F152 and K155, plays a critical role in CNTF receptor binding.
- Chicken CNTF receptors exhibit stricter recognition of the D1 cap region compared to rat receptors.
- Engineered antagonistic CNTF mutants offer potential tools for studying neurotrophic factor signaling and developing novel therapeutics.