Characterization of MdpS: an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis

Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin\u27s ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology

Salivary muc7 is a major carrier of blood group i type o-linked oligosaccharides serving as the scaffold for sialyl lewis x

Isolation of salivary MUC7 with gel electrophoresis allowed analysis by LC-MS and LC-MS(2) of released O-linked oligosaccharides and a thorough description of the glycosylation of this molecule, where high-molecular-weight oligosaccharides up to the size of 2790 Da and with up to three sialic acid residues were identified. A common theme of these novel high abundant oligosaccharides on MUC7 showed that the C-3 branch of the oligosaccharides consisted of branched I-antigen type structural epitopes (GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-), where the branch point was initiated on core 1 and core 2 galactose residues, and the branches were terminated by sialyl type 2 and sialyl Lewis x epitopes. Six sulfated sialylated oligosaccharides of low intensity were also identified, with the sulfate mainly on N-acetyl glucosamine residues located close to the reducing termini. One of these oligosaccharides was identified as a candidate for the high-affinity l-selectin ligand 6\u27-sulfo sialyl Lewis x. Neutral oligosaccharides and blood group antigens were found to be less abundant on MUC7 and the glycosylation appeared to be more preserved between individuals as compared to salivary MUC5B. This was illustrated by comparing the LC-MS spectra of MUC7 and MUC5B glycans from secretors (23 individuals) and nonsecretors (6 individuals). The data show that MUC7 provides a multivalent scaffold for sialylation, meeting the requirement for high-avidity binding via its glycosylation and mediator of the interaction between immune cells such as salivary neutrophils and oral bacteria

MUC5B glycosylation in human saliva reflects blood group and secretor status

This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from individuals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis (SDS–AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive β-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O individuals into larger population (29 individuals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 individuals. The remaining six individuals were characterized by a high degree of sialylation. These individuals were assigned as nonsecretors, whereas blood group-expressing individuals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Lea) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between individuals and that some of this variation is owing to blood group and secretor status.14 page(s

Characterization of MdpS : an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis

Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field