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Circularly permuted interleukin 4 retains proliferative and binding activity

R J Kreitman1, R K Puri, P McPhie

  • 1Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Cytokine
|May 1, 1995
PubMed
Summary

Circularly permuted interleukin-4 (IL-4) mutants retain significant binding and biological activity, demonstrating that free amino and carboxyl termini are not essential for IL-4 receptor interaction.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Human interleukin-4 (IL-4) is a 129-amino acid hormone crucial for immune responses.
  • The carboxyl and amino termini of IL-4 are spatially close and hypothesized to be vital for IL-4 receptor (IL-4r) binding.

Purpose of the Study:

  • To investigate the role of IL-4's native termini in receptor binding and biological activity.
  • To construct and characterize circularly permuted IL-4 mutants.

Main Methods:

  • Construction of circularly permuted IL-4 mutants (IL-4(38-37) and IL-4(105-104)) using a Gly-Gly-Asn-Gly-Gly linker.
  • Purification of mutant proteins from E. coli.
  • Assessment of binding affinity to IL-4r, proliferative activity, and CD23 upregulation.
  • Circular dichroism spectroscopy to evaluate tertiary structure.

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Main Results:

  • Purified IL-4 mutants IL-4(38-37) and IL-4(105-104) exhibited 50-100% of native IL-4's binding and proliferative activity.
  • Mutants successfully upregulated CD23 on Burkitt's lymphoma cells.
  • Circular dichroism confirmed retention of tertiary structures, with IL-4(38-37) closely resembling native IL-4.

Conclusions:

  • The native amino and carboxyl termini of IL-4 are not strictly required for optimal receptor binding and biological function.
  • Circularly permuted IL-4 variants maintain structural integrity and biological activity, offering insights into protein folding and receptor interactions.