Deciphering putatively transmembrane and secreted human gut microbial prolyl endopeptidases: therapeutic implications for celiac disease

Abstract

Celiac disease (CeD) is a chronic immune-mediated enteropathy triggered by the ingestion of gluten, a storage protein complex found in wheat, rye, and barley. Gluten comprises glutenin and gliadins, which are rich in proline and glutamine residues. The high proline content makes these proteins resistant to degradation by human gastrointestinal and brush-border proteases. While a strict gluten-free diet is currently the primary treatment for CeD, maintaining such a diet is often challenging and can significantly impact patients' quality of life. Therefore, alternative or supplementary therapeutic approaches are urgently needed. Certain gut-associated microbes have been reported to produce Prolyl Endopeptidases (PEPs), which can hydrolyze gluten peptides. In this study, we screened 10,903 bacterial PEPs from diverse taxa including Firmicutes, Bacteroidetes, Proteobacteria, Cyanobacteria, Fungi, and Animals. Using HMMER, these sequences were compared against curated reference databases comprising 115,692 transmembrane proteins and 40,344 secreted proteins from 169 gut-associated species. This analysis identified 372 transmembrane and 1,250 secreted PEP homologs. Among them, 31 transmembrane and 36 secreted PEPs from animal-associated species were selected for further analysis due to their closer similarity to human enzymes. The PEP from Sphingomonas capsulata—a component of the oral enzyme therapy latiglutenase—was used as a reference. Through comparative sequence and domain analysis, structural modeling, and molecular docking, ten PEPs (4 transmembrane and 6 secreted) were identified with high structural and functional similarity. Docking studies showed strong binding affinity to gluten peptides, with scores ranging from −7.2 to −10.2 kcal/mol. Furthermore, a prevalence analysis using five publicly available gut microbiome datasets comprising 414 samples (CeD and controls) revealed that the significant species harboring these enzymes were notably less abundant in CeD patients. This suggests a potential association between disease progression and the depletion of gluten-degrading microbial species. Together, our findings highlight a set of microbial enzymes with substantial gluten- degrading potential, which are underrepresented in individuals with Celiac disease. These enzymes present promising candidates for the development of next-generation enzyme-based therapies for CeD management.