N-terminal modified coix seed peptides: enhanced transepithelial transport in Caco-2 cell monolayers and modulates key targets in T2DM

LPFYPN, a novel hypoglycaemic peptide found in Coix seeds, inhibits Dipeptidyl peptidase IV (DPP-IV). This study explored how N-terminal chemical modification impacts LPFYPN's inhibitory activity, type, mechanism, cellular bioavailability, degradation, absorption, and transport using simulated Caco-2 cell monolayer models. Results showed LPFYPN was fully absorbed and utilized by Caco-2 monolayers, but its permeability represented only 8.8 % of the initial concentration. LPFYPN was mainly degraded in the intestine, creating LPFYP, PFYPN, and PFYPD peptides, which facilitated transmembrane transport via paracellular diffusion. Multidrug-resistant proteins mediated PFYPD's efflux absorption. The primary reasons for the limited inhibitory activity of the LPFYPN are the weaker interaction between its degradation products and DPP-IV, as well as the reduction in tight binding sites with the enzyme. N-terminal modifications (Methoxy polyethylene glycol carboxyl – LPFYPN and Selenocysteine - LPFYPN，mPEG-LPFYPN and SeCys-LPFYPN) improved DPP-IV inhibitory activity, with IC 50 values of 72.59 μM and 57.29 μM respectively. However, acetylated modified peptides (Ac-LPFYPN) decreased, with an IC 50 value of 104.43 μM. However, absorption of the three modified peptides improved significantly, with the apparent permeability coefficient (Papp) increasing by 1.15- to 2.53-fold, and better regulation of DPP-IV and GLP-1 protein levels. Ac-LPFYPN's transport mechanism mirrored parent peptides. SeCys-LPFYPN was involved in endocytosis as a transport pathway, and mPEG-LPFYPN's main transport mechanism was endocytosis-mediated, with P-glycoprotein mediating efflux. • N-terminal modification enhances the transport and absorption of LPFYPN in Caco-2 cell monolayers. • Modified peptides upregulated GLP-1 and downregulated DPP-IV protein levels dose-dependently. • mPEG- and SeCys-modifications improved DPP-IV inhibitory activity; Ac-reduced it. • LPFYPN was degraded into LPFYP, PFYPN, and PFYPD within the Caco-2 cell monolayers.