Reduction of biological activity of murine recombinant interleukin-1 beta by selective deamidation at asparagine-149

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Murine recombinant interleukin-1 beta (mIL-1 beta) is a key cytokine involved in immune responses.
• Recombinant proteins expressed in Escherichia coli can exhibit heterogeneity in post-translational modifications.
• Understanding these modifications is crucial for interpreting biological activity.

Purpose of the Study:

• To characterize the different forms of biologically active murine recombinant interleukin-1 beta (mIL-1 beta) produced in Escherichia coli.
• To investigate the structural modification responsible for the observed differences in mIL-1 beta forms.
• To assess the impact of this modification on mIL-1 beta's biological activity.

Main Methods:

• Purification of recombinant mIL-1 beta from Escherichia coli cell lysates.
• Isoelectric focusing (IEF) to separate protein isoforms based on pI.
• Chemical treatment (Tris buffer, pH 8.5, 37°C) to induce modification.
• Assays for co-mitogenic activity and receptor binding affinity.

Main Results:

• Two forms of mIL-1 beta were identified with distinct isoelectric points (pI 8.7 and pI 8.1).
• Treatment under specific conditions converted the pI 8.7 form to the pI 8.1 form.
• This conversion was attributed to the selective deamidation of asparagine residue at position 149 (Asn149).
• The deamidated form (pI 8.1) exhibited significantly lower co-mitogenic activity and receptor affinity (3- to 5-fold reduction).

Conclusions:

• The heterogeneity in recombinant mIL-1 beta observed is due to deamidation of Asn149.
• Deamidation of Asn149 critically impairs the biological functions of mIL-1 beta, including co-mitogenic activity and receptor binding.
• This finding has implications for the production and application of recombinant mIL-1 beta in research and therapeutic contexts.