Cathepsin D Drives the Formation of Hybrid Insulin Peptides Relevant to the Pathogenesis of Type 1 Diabetes

Abstract

Hybrid insulin peptides (HIPs) form in pancreatic β-cells through the formation of peptide bonds between proinsulin fragments and other peptides. HIPs have been identified in pancreatic islets by mass spectrometry and are targeted by CD4 T cells in patients with type 1 diabetes (T1D) as well as by pathogenic CD4 T-cell clones in nonobese diabetic (NOD) mice. The mechanism of HIP formation is currently poorly understood; however, it is well established that proteases can drive the formation of new peptide bonds in a side reaction during peptide bond hydrolysis. Here, we used a proteomic strategy on enriched insulin granules and identified cathepsin D (CatD) as the primary protease driving the specific formation of HIPs targeted by disease-relevant CD4 T cells in T1D. We also established that NOD islets deficient in cathepsin L (CatL), another protease implicated in the formation of disease-relevant HIPs, contain elevated levels of HIPs, indicating a role for CatL in the proteolytic degradation of HIPs. In summary, our data suggest that CatD may be a therapeutic target in efforts to prevent or slow the autoimmune destruction of β-cells mediated by HIP-reactive CD4 T cells in T1D.

Figure 1

Identification of HIP-forming proteases in β-cell lysates. A: Enriched insulin secretory granules were fractionated by SEC. Mass spectrometric analyses of the fractions led to the identification of 30 distinct proteases. B: Schematic of a proteolytic transpeptidation reaction leading to the formation of 2.5HIP between Des-(27-31)C-peptide and the ChgA fragment WQ6. C: BDC-2.5 T-cell responses to fractions incubated with or without 2.5HIP precursor peptides in the presence and absence of various inhibitors. See control experiments in Supplementary Fig. 7A. Experiment was done in triplicate. Above data show one representative experiment.

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.

Figure 3

Cleavage and transpeptidation site-specificity of human (h)CatD. A: E2 T-cell responses to in vitro reactions (pH 4.0–5.5) of HIP11 precursor peptide (C-peptide) incubated for 24 h in the presence of CatD. Results shown are the mean ± SEM from three independent experiments. Data were normalized to maximal IFN-γ signal for each experiment. See control experiments in Supplementary Fig. 7C. B: Incubation for 24 h of AspN-digested synthetic HIP11 sequence at pH 4.0 in presence and absence of 225 nmol/L human CatD. C: Mirror plot of synthetic HIP11 and HIP11 that formed in the presence of CatD and C-peptide. Data show the y4 ion of Des-(27-31)C-peptide that coisolated with the HIP11 ion during LC-MS/MS analyses. m/z, mass-to-charge. D: Following 24-h treatment of human C-peptide with variable concentrations of human (h)CatD, the spectral intensities of peptides resulting from cleavages at individual peptide bonds were summarized and graphed.

Figure 4

CatL does not contribute significantly to HIP formation in murine islets. A: BDC-2.5 T-cell responses to in vitro reactions (pH 5.0) of 2.5HIP precursor peptides incubated for 24 h in the presence and absence of various cysteine proteases and CatD. Responses of BDC-2.5 (B) and BDC-9.3 (C) T-cell clones to dispersed islets from NOD, NOD.CatL^+/−, and NOD.CatL^−/− islets. KO, knockout. See control experiments in Supplementary Fig. 7D and E. Results from A, B, and C are the mean ± SEM from three independent experiments. Data were normalized to the maximal IFN-γ signal for each experiment. D–F: Mirror plots showing the fragmentation spectra of the AspN-digested 2.5HIP, 6.9HIP, and murine HIP11 sequences identified in NOD CatL^−/− islets compared with the synthetic peptides. Presence of 2.5HIP, 6.9HIP, and mouse HIP11 in CatL-KO islets was fully validated through P-VIS (19).