Characterization of Novel Dipeptidyl Peptidase-IV Inhibitory Peptides from Soft-Shelled Turtle Yolk Hydrolysate Using Orthogonal Bioassay-Guided Fractionations Coupled with In Vitro and In Silico Study

Abstract

Five novel peptides (LPLF, WLQL, LPSW, VPGLAL, and LVGLPL) bearing dipeptidyl peptidase IV (DPP-IV) inhibitory activities were identified from the gastrointestinal enzymatic hydrolysate of soft-shelled turtle yolk (SSTY) proteins. Peptides were isolated separately using reversed-phase (RP) chromatography in parallel with off-line strong cation exchange (SCX) chromatography followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine sequences. Among these peptides, LPSW showed the highest DPP-IV inhibitory activity with an IC₅₀ value of 269.7 ± 15.91 µM. The results of the pre-incubation experiment and the kinetic study of these peptides indicated that WLQL is a true inhibitor and its inhibition toward DPP-IV is of an uncompetitive model, while LPLF, LPSW, and VPGLAL are real-substrates and competitive inhibitors against DPP-IV. The DPP-IV inhibitory peptides derived from SSTY hydrolysate in study are promising in the management of hyperglycemia in Type 2 diabetes.

Keywords: DPP-IV inhibitory peptides; LC-MS/MS; bioassay-guided fractionation; in silico analysis; soft-shelled turtle yolk protein.

Figure A1

IC₅₀ values of (A) LPSW, (B) VPGLAL, (C) WLQL, and (D) LPLF on DPP-IV.

Figure A2

(A) LC-MS profile of LPLF pre-incubated with DPP-IV at 37 °C for 3 h. (B) MS spectrum of fragment LF (m/z 279.12), and (C) MS spectrum of fragment LP (m/z 229.14) derived from LPLF pre-incubated with DPP-IV at 37 °C for 3 h.

Figure A3

(A) LC-MS profile of VPGLAL pre-incubated with DPP-IV at 37 °C for 3 h. (B) The MS spectrum of fragment GLAL (m/z 373.27), and (C) the MS spectrum of fragment VP (m/z 214.95) derived from VPGLAL after pre-incubation with DPP-IV.

Figure A4

(A) LC–MS chromatograms of SCX S2, RP R13, and the synthetic peptide LPLF, (B) MS spectra of LPLF originated from fraction SCX S2 (m/z 489.12, t_R 13.51 min), fraction RP R13 (m/z 489.08, t_R 13.51) and synthetic LPLF (m/z 489.07, t_R 13.59).

Figure A5

(A) LC–MS chromatograms of SCX S2, RP R13, and the synthetic peptide WLQL, (B) MS spectra of WLQL originated from fraction SCX S2 (m/z 559.07, t_R 13.51 min), fraction RP R13 (m/z 559.24, t_R 13.47) and synthetic WLQL (m/z 559.08, t_R 13.54).

Figure A6

(A) LC–MS chromatograms of SCX S2, RP R13, and the synthetic peptide LVGLPL, (B) MS spectra of LVGLPL originated from fraction SCX S2 (m/z 611.19, tR 13.71 min), fraction RP R13 (m/z 611.19, tR 13.69) and synthetic LVGLPL (m/z 611.19, tR 13.79).

Figure A7

(A) LC–MS chromatograms of SCX S2, RP R7, and the synthetic peptide LPSW, (B) MS spectra of LPSW originated from fraction SCX S2 (m/z 502.04, t_R 12.63 min), fraction RP R7 (m/z 502.06, t_R 12.61) and synthetic LPSW (m/z 502.06, t_R 12.65).

Figure A8

(A) LC–MS chromatograms of SCX S2, RP R7, and the synthetic peptide VPGLAL, (B) MS spectra of VPGLAL originated from fraction SCX S2 (m/z 569.05, t_R 12.67 min), fraction RP R7 (m/z 569.11, t_R 12.70) and synthetic VPGLAL (m/z 569.12, t_R 12.84).

Figure 1

(A) DPP-IV inhibitory activity with soft shelled turtle yolk (SSTY) hydrolysates by trypsin, pepsin, α-chymotrypsin, and GI (pepsin, trypsin and α-chymotrypsin) enzyme, respectively. Different letters (a–f) indicate significant differences between samples (p < 0.05). (B) SDS-PAGE analysis of SSTY protein (Lane 1), trypsin digest (Lane 2), pepsin digest (Lane 3), α-chymotrypsin digest (Lane 4), thermolysin digest (Lane 5), and GI hydrolysate (Lane 6) of SSTY proteins at concentration 50 µg/µL. M represents the molecular weight of the protein markers.

Figure 2

(A) Reverse-phase HPLC chromatogram with 10 mm × 250 mm, 5 µm column of turtle yolk hydrolysate. (B) DPP IV—Inhibition activity of RP-HPLC fractions (R1–R17). (C) DPP-IV inhibition activity of SCX fractions (S1–S10). Different letters (a–g) indicate significant differences between samples (p < 0.05).

Figure 3

(A) The distribution of five overlapping peptides in LC-MS chromatogram of SCX-S2 fraction; (B) MS spectra of LPLF (m/z 489.31 at t_R = 32.31 min), LPSW (m/z 502.27 at t_R = 29 min), VPGLAL (m/z 569.37 at t_R = 30.31 min), WLQL (m/z 559.32 at t_R = 33.05 min), and LVGLPL (m/z 611.41 at t_R = 35.30 min); (C) MS/MS spectra of the peptide with m/z 489.31, 502.27, 569.37, 559.32, and 611.41, respectively. The b- and y-series ions in the MS/MS spectra indicate the fragment ions of the peptides.

Figure 3

(A) The distribution of five overlapping peptides in LC-MS chromatogram of SCX-S2 fraction; (B) MS spectra of LPLF (m/z 489.31 at t_R = 32.31 min), LPSW (m/z 502.27 at t_R = 29 min), VPGLAL (m/z 569.37 at t_R = 30.31 min), WLQL (m/z 559.32 at t_R = 33.05 min), and LVGLPL (m/z 611.41 at t_R = 35.30 min); (C) MS/MS spectra of the peptide with m/z 489.31, 502.27, 569.37, 559.32, and 611.41, respectively. The b- and y-series ions in the MS/MS spectra indicate the fragment ions of the peptides.

Figure 4

LC-MS chromatogram of (A) LPSW (m/z 502) pre-incubated with DPP-IV for 3 h at 37 °C. (B) MS spectra of SW fragment (m/z 292.12), and (C) LP fragment (m/z 229.25). SW and LP were fragments derived from LPSW after 3 h pre-incubation with DPP-IV.

Figure 5

Pre-incubation experiment of LPLF, VPGLAL, WLQL, and LPSW. The error bars represent the standard deviation. Different letters (a–g) indicate significant differences between samples (p < 0.05). The concentration for each peptide is 500 µM.

Figure 6

Lineweaver–Burk double reciprocal plots for dipeptidyl peptidase IV (DPP-IV) inhibition with (A) LPLF (competitive), (B) LPSW (competitive), (C) VPGLAL (competitive), and (D) WLQL (uncompetitive) were determined at concentrations corresponding to their IC₅₀.