Purification, Identification and Molecular Docking of Immunomodulatory Peptides from the Heads of Litopenaeus vannamei

Abstract

In order to realize the high-value utilization of Litopenaeus vannamei (L. vannamei) heads, immunomodulatory peptides were prepared from the enzymatic hydrolysate of L. vannamei heads, and the action mechanism of immunomodulatory peptides was determined by molecular docking. The results showed that six proteases were used to hydrolyze L. vannamei head proteins, with the animal protease hydrolysate exhibiting the highest macrophage relative proliferation rate (MRPR). The enzymatic products were then sequentially purified by ultrafiltration, Sephadex G-15 gel chromatography, identified by liquid chromatography-mass spectrometry (LC-MS/MS), and finally selected for six immunomodulatory peptides (PSPFPYFT, SAGFPEGF, GPQGPPGH, QGF, PGMR, and WQR). These peptides maintained good immune activity under heat treatment, pH treatment, and in vitro gastrointestinal digestion. Molecular docking analysis indicated that these peptides showed great binding to both toll-like receptor 2 and 4 (TLR2 and TLR4/MD-2), leading to immunomodulation. The discarded L. vannamei heads in this article are considered to be promising food-borne immunomodulators that contribute to enhancing the immune function of the body.

Keywords: L. vannamei heads; identification; immunomodulatory peptides; molecular docking; purification.

Figure 1

Relative proliferation rate of macrophages (MRPR) and degree of hydrolysis (DH) with different enzymatic hydrolysis products (a), and molecular weight distribution of different enzymatic hydrolysates (b). Different uppercase letters in the figure indicate significant differences in DH (p < 0.05); different lowercase letters indicate significant differences in MRPR (p < 0.05).

Figure 2

MRPR in each ultrafiltration fraction (100 μg/mL) (a); Sephadex G-15 gel chromatogram (b), and MRPR in each component (c). Different lowercase letters indicate significant differences in MRPR (p < 0.05).

Figure 3

Evaluation of the immunomodulatory property of the M4-1 fraction. Effects of different concentrations of M4-1 components on phagocytosis of neutral red (a), NO release (b), and cytokines TNF-α (c), IL-2 (d), IL-6 (e), and IL-1β (f) in macrophages. Values in columns a–d with different superscripts indicate significant differences (p < 0.05).

Figure 4

MS/MS spectra of six immunomodulatory peptides identified and screened from the M4-1 fraction PSPFPYFT (a), SAGFPEGF (b), GPQGPPGH (c), QGF (d), PGMR (e), and WQR (f).

Figure 5

Stability analysis of synthetic peptides. Effects of gastrointestinal digestion (a), heat treatment (b), and pH treatment (c) on the relative proliferation rate of macrophages. Values in columns a–d with different superscripts indicate significant differences (p < 0.05).

Figure 6

Binding and interacting residues of peptides with TLR2 (ID: 1FYW). PSPFPYFT-TLR2 (a), GPQGPPGH-TLR2 (b), PGMR-TLR2 (c), and WQR-TLR2 (d). In the figure, green represents hydrogen bonds, yellow represents polypeptide ligands, and purple represents amino acid residues related to the action.

Figure 7

Binding and interacting residues of peptides with TLR4/MD-2 (ID: 5IJD). PSPFPYFT-TLR4/MD-2 (a), GPQGPPGH-TLR4/MD-2 (b), PGMR-TLR4/MD-2 (c), and WQR-TLR4/MD-2 (d). In the figure, green represents hydrogen bonds, yellow represents polypeptide ligands, and purple represents amino acid residues related to the action.