Four cysteine residues contribute to homodimerization of chicken interleukin-2

Authors: DENG, CHENG - Jilin University; TAN, HAILIANG - Jilin University; ZHOU, HONGDA - Jilin University; WANG, MENGYUN - Jilin University; LU, YAN - Jilin University; XU, JIACUI - Jilin University; Zhang, Huanmin; HAN, LIMEI - Shenyang Agricultural University; AI, YONGXING - Jilin University

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/12/2019
Publication Date: 11/15/2019
Publication URL: https://handle.nal.usda.gov/10113/6764847
Citation: Deng, C., Tan, H., Zhou, H., Wang, M., Lu, Y., Xu, J., Zhang, H., Han, L., Ai, Y. 2019. Four cysteine residues contribute to homodimerization of chicken interleukin-2. International Journal of Molecular Sciences. 20(22):5744. https://www.doi.org/10.3390/ijms20225744.
DOI: https://doi.org/10.3390/ijms20225744

Interpretive Summary: Interleukin-2 is a member of interleukin, a type of cytokine signaling molecule in the immune system. A series of experiments has been conducted, which identified four amino acid substitutes in the chicken linterleukin-2 protein. The interesting thing is that all four substitutes were found necessary in a functional intervention of the pleiotropic cytokine molecules. Lack of any one of the four substitutes will be insufficient to insert such function. This finding advanced the basic understanding on cytokine functionality and its molecular entity and could potentially facilitates future investigation on the cytokine variants and its biological functions.

Technical Abstract: Interleukin-2 (IL-2) is a pleiotropic cytokine regulating immune and nervous systems. Mammalian and bird IL-2s have different protein sequences but perform similar functions. In this study, two bands were detected by immunoblotting using an antibody against freshly purified chicken IL-2 (chIL-2). The molecular weight of the larger band was approximately twice as that of the chIL-2 monomer, although a chIL-2 complex or homodimer has never been reported. To explain this intriguing result, several dissociation agents were used to examine the intermolecular forces between components of the proposed chIL-2 complex. It was found that intermolecular disulfide bond promoting homodimerization of chIL-2. Subsequently, mutation of Cys residues of chIL-2 revealed that mutation of all four Cys residues disrupted homodimerization, but a single, dual, or triple Cys mutation failed to disrupt homodimerization, suggesting that all four Cys residues on chIL-2 contribute to this dimerization. Functional analysis showed that both monomeric and dimeric chIL-2 consisting of either wild type or mutant chIL-2 were able to stimulate the proliferation of CD4+ T cell. Overall, this study revealed that the recombinant chIL-2 purified from either E. coli or Spodoptera frugiperda (Sf9) cells could homodimerize in vitro, with all four Cys residues on each chIL-2 protein contributing to this homodimerization, and dimerization and Cys mutation not impacting chIL-2 induced stimulation of chicken CD4+ T cells.