GAPDH Released from Lactobacillus johnsonii MG Enhances Barrier Function by Upregulating Genes Associated with Tight...

Extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple interactions with various gut epithelial components. For instance, GAPDH in Lactobacillus johnsonii MG cells interacts with junctional adhesion molecule-2 (JAM-2) in Caco-2 cells and enhances tight junctions. However, the specificity of GAPDH toward JAM-2 and its role in the tight junctions in Caco-2 cells remain unclear. In the present study, we assessed the effect of GAPDH on tight junction regeneration and explored the GAPDH peptide fragments required for interaction with JAM-2. GAPDH was specifically bound to JAM-2 and rescued H₂O₂-damaged tight junctions in Caco-2 cells, with various genes being upregulated in the tight junctions. To understand the specific amino acid sequence of GAPDH that interacts with JAM-2, peptides interacting with JAM-2 and L. johnsonii MG cells were purified using HPLC and predicted using TOF-MS analysis. Two peptides, namely ¹¹GRIGRLAF¹⁸ at the N-terminus and ³²³SFTCQMVRTLLKFATL³³⁸ at the C-terminus, displayed good interactions and docking with JAM-2. In contrast, the long peptide ⁵²DSTHGTFNHEVSATDDSIVVDGKKYRVYAEPQAQNIPW⁸⁹ was predicted to bind to the bacterial cell surface. Overall, we revealed a novel role of GAPDH purified from L. johnsonii MG in promoting the regeneration of damaged tight junctions and identified the specific sequences of GAPDH involved in JAM-2 binding and MG cell interaction.