Role of salivary microbial enzymes and proline-rich proteins in celiac disease
INTRODUCTION: Human saliva contains a variety of microorganisms and salivary proteins implicated in oral health and disease. The oral bacterium R. mucilaginosa harbors glutamine endoprotease activity degrading salivary proline-rich proteins (PRPs) rich in glutamine residues. PRPs share structural features with dietary gluten proteins, which trigger celiac disease (CD). Their structural similarities and shared destination of the gastrointestinal tract raise the potential for the involvement of PRPs in CD pathogenesis. The aims of this study were to: 1) Investigate to what extent R. mucilaginosa cell-associated enzymes degrade gluten and abolish their immunogenicity; 2) Compare gluten-degrading enzyme activities and microbiomes in whole saliva (WS) from healthy and CD subjects; 3) Study the potential immunogenicity of salivary PRPs. METHODS: Studies on gluten degradation by R. mucilaginosa comprised SDS-PAGE, RP-HPLC, LC-ESI-MS/MS, and ELISA. Clinical studies were conducted with healthy and CD patient groups. Salivary hydrolytic activities were assessed towards Z-YPQ-pNA and gliadin-derived immunogenic 33-mer/26-mer peptides. Oral microbiomes were analyzed by 16S rDNA sequencing. Induction of cytokines (TNF-alpha, IL-10, IFN-gamma, and IL-21) by PRPs was studied in peripheral blood mononuclear cells (PBMCs) collected from CD patients. RESULTS: R. mucilaginosa cell-associated enzymes degraded gliadins/33-mer/26-mer, decreased their recognition by TG2 and abolished epitopes recognized by R5 antibody. WS showed no differences between healthy and CD patients with regard to activities relevant in gluten degradation, and salivary microbiome compositions were similar. PRPs protein patterns revealed minor differences that were not group-specific. Despite structural similarities, PRPs did not stimulate cytokines production by PBMCs, nor did they compete with gliadin-induced cytokine secretion. CONCLUSION: From a therapeutic view point, R. mucilaginosa cells and/or their gluten-degrading enzymes may offer novel perspectives for CD treatment. From an oral physiological perspective, endogenous WS gluten-degrading activities were low and comparable between healthy and CD groups, suggesting such activities may not be sufficient for gluten digestion in vivo, and further supporting the dietary supplementation concept. PRPs do not seem to harbor gliadin-like elements relevant in CD pathogenesis. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs is of interest to inform development of gluten proteins lacking immunogenic epitopes, and their design is discussed.