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Structure-function analysis of the p35 subunit of mouse interleukin 12
J J Zou1, D S Schoenhaut, D M Carvajal
1Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110-1199.
The Journal of Biological Chemistry
|March 17, 1995
Summary
Mouse and human Interleukin-12 (IL-12) exhibit species-specific activity due to the p35 subunit. Key residues in mouse p35 are crucial for receptor interaction and signaling, explaining IL-12
Area of Science:
- Immunology
- Molecular Biology
- Protein Engineering
Background:
- Interleukin-12 (IL-12) is a cytokine with distinct species-specific activity.
- Mouse IL-12 functions on both mouse and human cells, whereas human IL-12 is specific to human cells.
- The p35 subunit of the IL-12 heterodimer dictates this species specificity.
Purpose of the Study:
- To identify the specific regions on the mouse p35 subunit that interact with the mouse IL-12 receptor.
- To elucidate the role of these regions in receptor binding and downstream signaling.
- To understand the molecular basis of Interleukin-12 (IL-12) species specificity.
Main Methods:
- Construction of mouse-human chimeric p35 molecules by exchanging nonhomologous sequences.
- Assessment of bioactivity and competitive binding of mutant IL-12 heterodimers on mouse cells.
- Site-directed mutagenesis to pinpoint critical residues within the p35 subunit.
Main Results:
- A chimeric p35 molecule with five residue changes in three discontinuous sites showed a 750-3000 fold reduction in bioactivity on mouse cells.
- Competitive binding affinity was reduced only 30-fold for the same mutant IL-12 on mouse cells.
- These findings indicate that the identified residues significantly impact signaling more than receptor binding.
Conclusions:
- The mouse p35 subunit is involved in both IL-12 receptor binding and signal transduction.
- Specific mutations in mouse p35 drastically impair signaling while having a moderate effect on receptor binding.
- Five identified residues on mouse p35 are essential for interaction with the mouse IL-12 receptor, contributing significantly to species specificity.