Characterization of a highly conserved MUC5B-degrading protease, MdpL, from Limosilactobacillus fermentum

**Background:** MUC5B is the predominant gel-forming mucin in the oral cavity, respiratory tract, and endocervix, constituting the main nutrient source for oral bacteria. Limosilactobacillus fermentum is a common member of the oral microbiome, but its role in mucin degradation and biofilm dynamics is debated. A previous study identified a putative O-sialoglycoprotein endopeptidase (OSGP) upregulated 2.7-fold when L. fermentum was grown in a MUC5B environment. This study characterizes that enzyme, renamed MdpL (Mucin Degrading Protease L), from L. fermentum.

**Methods:** MdpL (GenBank WP_057727250.1) was recombinantly expressed in E. coli as a His-tagged fusion protein, purified from inclusion bodies, and refolded. Physicochemical characterization assessed pH (4.5–8.0), temperature (29–45°C), cations/EDTA (2 mM CaCl₂, MgCl₂, ZnCl₂, EDTA), NaCl (0–500 mM), and reducing agents (L-cys 10–100 mM, DTT 10–100 mM) using Etanercept as substrate with SDS-PAGE and densitometric quantification. Protease inhibition was tested with 11 inhibitors. Substrate specificity was evaluated against casein, IgG subclasses (1,2,4), pIgA, Abatacept, Etanercept, and fetuin, with and without O-glycan removal. MUC5B degradation was analyzed by SDS-PAGE, Western blot, and LC–MS/MS. Peptide specificity was assessed using seven peptides (Drosocin, H2686, H8390, Amyloid β 10–20, Neuromedin U-25, MOG, Insulin Chain B Oxidized). Cellular localization was determined by LC–MS/MS of lysate, surface-associated (papain-digested), and extracellular fractions of L. fermentum culture.

**Key Results:** MdpL is highly conserved within L. fermentum (99.7% identity between strains KRN17993.1 and WP_057727250.1) and shows homology with Limosilactobacillus spp., Gardnerella vaginalis, and early biofilm colonizers including Streptococcus mutans. No MdpL homolog was found in Porphyromonas gingivalis, Helicobacter pylori, or Akkermansia muciniphila. MdpL activity peaked at pH 5.5–7.0, retaining 60% activity at pH 4.5 and 8.0. Optimal temperature was 37°C. ZnCl₂ (2 mM) completely inhibited activity, while CaCl₂, MgCl₂, and EDTA slightly increased it. Reducing agents (L-cys, DTT) enhanced activity at 10 mM. Complete inhibition was observed with AEBSF, ALLN, antipain, chymostatin, E64, leupeptin, and phosphoramidon; bestatin and PMSF showed moderate inhibition; aprotinin, EDTA, and pepstatin had no effect. MdpL hydrolyzed casein at ~30% of RgpB activity. It did not significantly cleave IgG subclasses or pIgA, but hydrolyzed Abatacept, Etanercept, and fetuin regardless of O-glycan presence. MUC5B was degraded after 24 h incubation, producing fragments <200 kDa. LC–MS/MS identified three unique MdpL-derived MUC5B peptides, with cleavage sites in the N-terminal and B domains. Peptide specificity analysis (89 high-score peptides) showed preference for hydrophobic amino acids at the P1' position, with a strong preference for substrates longer than 20 amino acids. MdpL was detected in lysate (10⁸ XIC intensity) and surface-associated (10⁷ XIC intensity) fractions, but not extracellularly.

**Clinical Implications:** MdpL represents a novel mucin-degrading protease from L. fermentum that acts independently of O-glycans, distinguishing it from previously characterized O-glycoproteases. Its conservation across oral commensals and activity at pH 5.5–7.0 (relevant to dental biofilm pH fluctuations) suggest a role in maintaining eubiotic oral environments through MUC5B degradation for nutrient acquisition. The absence of MdpL in periodontal pathogens like P. gingivalis supports a commensal rather than virulence-associated function. Understanding MdpL activity may inform probiotic strategies and oral microbiome modulation, though further studies are needed to clarify its role in multispecies biofilm dynamics and caries pathogenesis.