Rheumatoid arthritis, an autoimmune disease that causes inflammation around joints and damages ligaments and cartilage, spreads inflammation to the skin and blood vessels, causing headaches, anemia, and, in severe cases, vasculitis. This disease results from an imbalance between pro- and anti-inflammatory cytokines. Recent studies have found that truncated-Inhibitor K562 (tIK) protein suppresses the expression of inflammatory cytokines. Therefore, we conducted a study to develop a therapeutic candidate peptide using only the region expressing anti-inflammatory properties among the amino acid sequences of the tIK peptide. We investigated phosphorylation patterns in macrophages of tIK transgenic mice and found that tIK protein phosphorylates tyrosine 496th of the interleukin (IL)-10 receptor subunit alpha in the mice without inflammation. To find the specific amino acid sequence part of the tIK protein that caused phosphorylation of the IL-10 receptor, the structure of tIK was predicted by comparing it with IL-10 using sequence homology modeling, and as a result, four epitope candidate groups were determined. By comparing the anti-inflammatory activity of each epitope through the TH17 cell differentiation test, it was found that the 18 amino acid epitope had the best anti-inflammatory properties, and the peptide was named tIK-18mer. Based on this peptide, shorter anti-inflammatory 9-mers and 14-mers were also found.
Peptides uniformly labeled with 15N were expressed by culturing E. coli and the experimental purified procedure was optimized to analyze by MALDI-TOF and NMR. The secondary structure of the peptide was studied using CD and 2D NMR, and the interaction between the receptor and the tIK series was studied using chemical shift perturbation. Finally, an anti-inflammatory test was performed to confirm the anti-inflammatory effect of the purified peptide.