Fig 2: Characterization of CD26 expression, mDPP4 enzymatic activity, and cytokine expression in T helper cell lines. (A, B) Jurkat E6 and H9 Th1 cells were harvested and analyzed for the expression of T cell-specific surface markers, including CD3, CD4, and CD26 (A), and for their mDPP4 enzymatic activity (B). (C) H9 Th1 cells were treated with various T cell activators, including T-Activator CD3/CD28, PWM (10 µg/mL), and PMA (10 µg/mL) for 3 h. The expressions of 18 cytokines and 6 MMPs were measured in the culture supernatants of treated cells using two different panels of fluorescent multiplex bead assays. Activated T helper cell-specific cytokines, including IL-2, IL-10, IL-13, GM-CSF, IFN-γ, and TNF-α, are shown. VEGF and MMP 9 are also included as detected high in all the samples. (D-F) H9 Th1 cells were treated with 10 µg/mL PWM for 12 h and subjected to mDPP4 enzymatic assays (D), FACS analysis for T helper cell-specific surface markers such as CD3, CD4, CD26, and CD28 (E), and analysis for apoptosis using an annexin V kit and flow cytometry (F). All analyses were repeated in three independent batches, and the data are shown as the means and standard deviation. **p<0.005; ***p<0.001 (paired t-test).

Fig 3: Effects of diprotin A and berberine on mDPP4 enzymatic activity, cell profiles, and T helper-specific cytokine expression in H9 Th1 cells. (A) The chemical structures of diprotin A and berberine, representative DPP4 inhibitors, but not antidiabetic drugs, are shown. (B-D) Cell pellets and culture supernatants were harvested 12 h after treatment of H9 Th1 cells with diprotin A and berberine (dose range, 0-20 µg/mL) in the presence of 10 µg/mL PWM. The mDPP4 enzymatic activity of the cell pellets was determined (B). The cell profiles, including cell counts and viability, of the PWM-treated cells with or without DPP4 inhibitors were determined (C). The values of Th1-specific cytokines, including IL-2, IL-10, IL-13, GM-CSF, IFN-γ, and TNF-α, and VEGF in the culture supernatants were measured simultaneously (D). All analyses were the same as those described in Fig. 2.

Fig 4: Comparison of the inhibitory effects of various DPP4 inhibitors on rDPP4 and mDPP4 activities at different time points. The DPP4 inhibitory activities of evogliptin, sitagliptin, diprotin A, and berberine were measured using recombinant DPP4 (rDPP4) protein or cell pellets of H9 Th1 cells. DPBS or DMSO were used as a control. The recombinant DPP4 proteins were incubated with evogliptin and sitagliptin (2 ng/mL and 2 µg/mL) or diprotin A and berberine (20 and 50 µg/mL); harvested after 0, 0.5, 1, 3, 6, 12, and 24 h; and subjected to assays for measuring the DPP4 activity (A, C). The results of DPP4 enzymatic activity after 30 min of incubation are shown (B, D). The H9 Th1 cell pellets were incubated with evogliptin and sitagliptin (2 ng/mL and 2 µg/mL, respectively) or diprotin A and berberine (20 and 50 µg/mL); harvested after 0, 0.5, 1, and 3 h; and subjected to DPP4 assays (E, G). The results of DPP4 activity after 30 min incubation are shown (F, H). All analyses were performed in three independent experiments, and the data are represented as the means and standard deviation. *p<0.05; **p<0.005; and ***p<0.001 (paired t-test).

Fig 5: Models built on product peptides depicting DPP4 cleavage specificity.(A) Bar plots showing the predictive power (variance explained for semi-tryptic (product) peptides) of a total of twelve linear models, each incorporating a specific set of predictors, indicated on the left and schematically displayed on the right. The red connecting line indicates interaction terms between the respective amino acids. A downward arrow indicates the cleavage site. The performance of each model is quantified via the adjusted R2 to account for differences in the number of parameters among the models. (B) Sequence logo representing the cleavage specificity of DPP4 inferred by a linear model considering semi-tryptic (product) peptides (A, bottom). For each amino acid at position P1, a sequence logo triplet depicts the cleavage specificity considering the contributions of P1 as well as the two neighboring (P2 and P1’) amino acids. The height of each amino acid letter in the sequence logo represents the contribution to cleavage efficiency. Positive numbers indicate an increase in cleavage efficiency while negative numbers denote a reduction in cleavage efficiency. The total efficiency of a triplet is given by the sum of the contributions of the amino acids occupying three (P2, P1, and P1’) positions. (C) Comparison of models generated using semi-tryptic peptides (DPP4 products; y axis) or tryptic peptides (input; x axis). Each dot represents one model parameter; Pearson correlation is indicated. (D) Scatter plot of experimentally determined peptide half-lives upon incubation with DPP4 (from (Keane et al, 2011)) vs. qPISA model score obtained by analyzing the semi-tryptic (product) peptides. Each dot is a unique peptide whose first three amino acids are indicated.