(P26-038-25) Peptidomics-Based Screening Approach for Identifying GPCR-Binding Peptide Ligands From Food Protein Hydrolysates

Objectives: Food-derived bioactive peptides (BPs) have received considerable attention as postbiotics for promoting gut health. Yet, most studies focus on the functional effects of crude extracts, with limited emphasis on identifying specific peptides and cellular targets. We previously demonstrated that probiotics-aided collagen peptide (CP) fractions repair intestinal epithelium via GPR35-mediated ERK1/2 signaling pathways in Caco-2 cells. Building on this, this study aims to develop a screening method to identify BP ligands and receptors by isolating receptor-ligand complexes.

Methods: For maximal GPR35 expression, Caco-2 cells were differentiated into monolayers and characterized for their functional and morphological resemblance to mature small intestinal (SI) enterocytes using qPCR and SEM. Differentiated Caco-2 monolayers were treated with CP fractions, and GPR35-CP complexes were isolated and enriched using co-IP. The sequences of bound collagen peptides were identified using LC-MS². Peptides were synthesized and validated for their functions using GPCR β-arrestin recruitment and cell migration assays.

Results: Differentiated Caco-2 monolayers showed upregulated gene expressions of differentiation markers and microvilli formation, demonstrating functional and morphological features of mature SI enterocytes. Under these conditions, GPR35 was significantly up-regulated at both the gene and protein levels compared to its endogenous levels. GPR35-binding CPs were identified, and they exhibited dose-dependent binding and activation of GPR35 as well as GPR35-mediated wound healing activities.

Conclusions: This study establishes a novel screening method for identifying BPs that target specific cellular receptors. These findings advance our understanding of peptide-receptor interactions and provide a foundation for targeted postbiotics for gut health.

Funding Sources: This research was supported by Basic Science Research Program and Bio & Medical Technology Development Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MSIT) (RS-2024-00413282 & RS-2021-NR056579), and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2023-KH141436).