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. 2022 Aug 30;71(12):2793–2803. doi: 10.2337/db22-0303

Figure 2.

Figure 2

Cleavage and transpeptidation site-specificity of murine (m)CatD. A: BDC-2.5 T-cell responses to in vitro reactions (pH 4.0–5.5) of 2.5HIP precursor peptides (insulin 2 C-peptide and WQ6) (see Table 1) incubated for 2 h in the presence and absence of mCatD. B: BDC-2.5 T-cell responses to precursor peptides incubated in the presence of mCatD (pH 4.0) for 0, 1, 2, 24, 48, and 72 h. Results from A and B are the mean ± SEM from three independent experiments. Data were normalized to the maximal IFN-γ signal for each experiment. See control experiments in Supplementary Fig. 7B. C: Incubation for 24 h of AspN-digested synthetic 2.5HIP sequence at pH 4.0 in the presence and absence of 75 nmol/L murine CatD. Following AspN digestion, the in vitro formation of 2.5HIP (D) and 2.5HIP-b (E) was validated using the P-VIS protocol (19). Figure shows mirror plots generated during peptide validation. F: Following 24-h treatment of murine C-peptide with variable concentrations of mCatD, the spectral intensities of peptides resulting from cleavages at individual peptide bonds were summarized and graphed.