Mechanism of intestinal paracellular absorption of shrimp peptide QMDDQ regulated by myosin light chain kinase.

Abstract

<h4>Background</h4>Myosin light chain kinase (MLCK) was identified as a key regulator of the paracellular absorption and transport of the shrimp peptide Gln-Met-Asp-Asp-Gln (QMDDQ).<h4>Results</h4>Molecular simulations revealed that QMDDQ formed a stable bond with MLCK through a hydrogen bond network, which ultimately facilitated its absorption. The concentration of absorbed QMDDQ in mouse blood after oral administration decreased from 128.91 ± 8.65 ng mL^-1 to 101.23 ± 4.61 ng mL^-1 (P < 0.05) in the presence of the MLCK-specific inhibitor ML-7. QMDDQ decreased the fluorescence intensity of the tight junctions (TJs) components zonula occludens-1 (ZO-1) and occludin, temporarily opened the mesh structure and up-regulated the expression of claudin-2. The fluorescence intensities of ZO-1 and occludin were inversely proportional to that of fluorescein isothiocyanate-labeled peptide (FQQ-5). QMDDQ reduced MLCK inhibition by ML-7 and decreased ZO-1 and occludin expression, opening TJs in the intestinal wall, which facilitated paracellular absorption of QMDDQ in vivo. Using a clustered regularly interspaced short palindromic repeats (i.e. CRISPR)/CRISPR-associated protein 9 (i.e. Cas9) system, we generated MLCK^-/- Caco-2 cells and showed with western blotting that QMDDQ up-regulated the expression of MLCK, MLC, p-MLC and claudin-2, as well as down-regulated the expression of ZO-1 and occludin. The increase in MLC phosphorylation following MLCK activation dynamically regulated TJs, widened intercellular gaps and enabled the paracellular absorption of QMDDQ.<h4>Conclusion</h4>Our results provide a theoretical basis for the explanation of efficient absorption of bioactive peptides, and provide new raw materials for the development of functional foods. © 2025 Society of Chemical Industry.