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

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.

Lactobacillus johnsonii enhances the gut barrier integrity via the interaction between GAPDH and the mouse tight junction protein JAM-2.

Commensal intestinal microbiota interacts with gut epithelial cells in the host by binding to specific host receptors. Several pattern recognition receptors on the gut that sense conserved microbial-associated molecular patterns have been reported; however, many of the gut receptor molecules involved in bacterial binding have not yet been identified. In this study, commensal intestinal bacteria interacting with mouse gut surface proteins were screened from fecal bacterial samples, to identify novel receptors on the epithelial cells in the mouse gut. Among the screened intestinal lactic acid bacteria, the frequently isolated Lactobacillus johnsonii MG was used for the purification of gut receptor proteins. An approximately 30 kDa protein was purified using affinity resin coupled surface layer proteins isolated from L. johnsonii MG. The purified gut protein was identified as a member of the tight junction protein family, junctional adhesion molecule-2 (JAM-2). As expected, the tight junctions of Caco-2 cells damaged by H2O2 were repaired by incubation with L. johnsonii MG. RNA sequence analysis showed significant upregulation of the expression of genes for tight junctions, anti-inflammatory effects, transcriptional regulation, and apoptosis in Caco-2 cells, following L. johnsonii MG treatment. In L. johnsonii MG, the surface layer 40 kDa protein was purified with gut protein-coupled affinity resin and identified as the moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These results suggest that L. johnsonii MG promotes the barrier function integrity in Caco-2 cells via GAPDH-JAM-2 binding. Here, we propose a promising approach to identify novel gut receptor molecules based on commensal bacterial interactions and understand host-bacterial communication in a mouse model.

Differentiation Between Chaenomelis Fructus and its Common Adulterant, Guangpi Mugua.

BACKGROUND: The dried fruit of Chaenomeles speciosa, known as Chaenomelis Fructus or Zhoupi Mugua, is a type of traditional Chinese medicine (TCM) that is widely used to treat many diseases. Guangpi Mugua, the dried fruit of the Chaenomeles sinensis, is its most commonly known adulterant. OBJECTIVE: To establish a robust approach for the quality control and identification of Chaenomelis Fructus. METHOD: Thin-layer chromatography (TLC) was optimized and used to discriminate Chaenomelis Fructus from Guangpi Mugua. High-performance liquid chromatography (HPLC) combined with fingerprint analysis and partial least-squares (PLS) discrimination analysis (DA) was employed to study the chemical differences between Chaenomelis Fructus and Guangpi Mugua. The single standard to determine multi-components (SSDMC) method, with credible precision, repeatability, stability, and durability, was developed for quantitative analysis of the abundant markers. RESULTS: The developed TLC and HPLC methods were effective in the authentication of Chaenomelis Fructus. Moreover, oleanolic acid, ursolic acid, pomolic acid, corosolic acid, 3-O-acetylpomolic acid, and one unknown compound were identified to be critical markers for the discrimination of Chaenomelis Fructus from Guangpi Mugua. CONCLUSION: Adulteration has always been a challenge in the development of TCM. This study presents useful insights that may help solve the problem of adulteration during the preparation of Chaenomelis Fructus. HIGHLIGHTS: The present study provides a systematic method for the quality control of Chaenomelis Fructus. This is therefore the first step towards solving the problem of adulteration to improve the clinical safety and effectiveness of Chaenomelis Fructus.