Dataset Information

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains.

ABSTRACT: The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins and the hemostasis protein von Willebrand factor. Here we report the x-ray crystal structure of the mucin 2 multimerization module, found to form a dimer linked by two intersubunit disulfide bonds. The dimer structure calls into question the current model for intestinal mucin assembly, which proposes disulfide-mediated trimerization of the same module. Key residues making interactions across the dimer interface are highly conserved in intestinal mucin orthologs, supporting the physiological relevance of the observed quaternary structure. With knowledge of the interface residues, it can be demonstrated that many of these amino acids are also present in other mucins and in von Willebrand factor, further indicating that the stable dimer arrangement reported herein is likely to be shared across this functionally broad protein family. The mucin 2 module structure thus reveals the manner by which both mucins and von Willebrand factor polymerize, drawing deep structural parallels between macromolecular assemblies critical to mucosal epithelia and the vasculature.

SUBMITTER: Javitt G

PROVIDER: S-EPMC6739602 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains.

Javitt Gabriel G Calvo María Luisa Gómez MLG Albert Lis L Reznik Nava N Ilani Tal T Diskin Ron R Fass Deborah D

Journal of molecular biology 20190713 19

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins ...[more]

PMID: 31310764

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Project description:The intermolecular interactions in concentrated solutions of pig submaxillary mucin (PSM) and sheep submaxillary mucin (SSM) were studied by mechanical spectroscopy. PSM and SSM were purified from detectable protein and nucleic acid by equilibrium centrifugation in a CsCl density gradient. PSM and SSM isolated in the presence of proteinase inhibitors showed distinct differences from preparations isolated in the presence of 0.2 M-NaCl alone, the latter having a carbohydrate and amino acid analysis similar to other preparations isolated by precipitation or ion-exchange techniques. Gel-filtration studies showed that preparations isolated in the presence of 0.2 M-NaCl alone were dissociated into smaller-sized glycoprotein units by 3.5 M-CsCl or 2.0 M-NaCl (SSM), pH 2.0 (PSM) or heating at 100 degrees C for 10 min (PSM and SSM). Preparations isolated in the presence of proteinase inhibitors were not dissociated by these treatments. Proteolysis fragmented all submaxillary mucin preparations into small glycopeptides of Mr 13,700 for PSM and of Mr 14,000 and 15,000 for SSM. PSM preparations when concentrated formed viscoelastic gels, as determined by mechanical spectroscopy. In contrast, SSM showed characteristics of a weak viscoelastic liquid under comparable conditions (coil overlap). PSM glycoprotein isolated in proteinase inhibitors formed weak viscoelastic gels at concentrations between 5 and 15 mg/ml. Preparations of PSM glycoprotein isolated in the presence of 0.2 M-NaCl (concentration 10-97 mg/ml) had the same overall mechanical gel structure as those preparations extracted in the presence of proteinase inhibitors. This gel structure was seen to collapse following proteolysis of both preparations or after acid treatment of the glycoprotein isolated in the presence of 0.2 M-NaCl, consistent with the breakdown in size of the polymeric glycoprotein. Treatment of PSM gel with 0.2 M-2-mercaptoethanol caused a surprising increase in gel strength, which was further markedly increased on removal of the reducing agent by dialysis. An association of reduced subunits of PSM was observed by gel filtration after removal of 0.2 M-2-mercaptoethanol. These results point to intermolecular disulphide exchange occurring on reduction of these PSM glycoprotein preparations. These results demonstrate that gel formation in PSM glycoprotein is similar to that for other gastrointestinal mucus glycoproteins from stomach to colon. Gel formation in PSM, as in other mucins, depends on polymerization of subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:Importance:Persistent, viscous middle ear effusion in pediatric otitis media (OM) contributes to increased likelihood of anesthesia and surgery, conductive hearing loss, and subsequent developmental delays. Biomarkers of effusion viscosity and hearing loss have not yet been identified despite the potential that such markers hold for targeted therapy and screening. Objective:To investigate the association of gel-forming mucins and aquaporin 5 (AQP5) gene expression with inflammation, effusion viscosity, and hearing loss in pediatric OM with effusion (OME). Design, Setting, and Participants:Case-control study of 31 pediatric patients (aged 6 months to 12 years) with OME undergoing tympanostomy tube placement and control individuals (aged 1 to 10 years) undergoing surgery for cochlear implantation from February 1, 2013, through November 30, 2014. Those with 1 or more episodes of OM in the previous 12 months, immunologic abnormality, anatomical or physiologic ear defect, OM-associated syndrome (ie, Down syndrome, cleft palate), chronic mastoiditis, or history of cholesteatoma were excluded from the study. All patients with OME and 1 control were recruited from Children's Hospital of Wisconsin, Milwaukee. The remainder of the controls were recruited from Sick Kids Hospital in Toronto, Ontario, Canada. Main Outcomes and Measures:Two to 3 middle ear biopsy specimens, effusions, and preoperative audiometric data (obtained <3 weeks before surgery) were collected from patients; only biopsy specimens were collected from controls. Expression of the mucin 2 (MUC2), mucin 5AC (MUC5AC), mucin 5B (MUC5B), and AQP5 genes were assayed in middle ear biopsy specimens by quantitative polymerase chain reaction. One middle ear biopsy specimen was sectioned for histopathologic analysis. Reduced specific viscosity of effusions was assayed using rheometry. Results:Of the 31 study participants, 24 patients had OME (mean [SD] age, 50.4 [31.9] months; 15 [62.5%] male; 16 [66.7%] white) and 7 acted as controls (mean [SD] age, 32.6 [24.4] months; 2 [26.6%] male; 6 [85.7%] white). Mucins and AQP5 gene expression were significantly higher in patients with OME relative to controls (MUC2: ratio, 127.6 [95% CI, 33.7-482.7]; MUC5AC: ratio, 3748.8 [95% CI, 558.1-25 178.4]; MUC5B: ratio, 471.1 [95% CI, 130.7-1697.4]; AQP5: ratio, 2.4 [95% CI, 1.1-5.6]). A 2-fold increase in MUC5B correlated with increased hearing loss (air-bone gap: 7.45 dB [95% CI, 2.65-12.24 dB]; sound field: 6.66 dB [95% CI, 6.63-6.69 dB]), effusion viscosity (2.75 mL/mg; 95% CI, 0.89-4.62 mL/mg), middle ear epithelial thickness (3.5 ?m; 95% CI, 1.96-5.13 ?m), and neutrophil infiltration (odds ratio, 1.7; 95% CI, 1.07-2.72). A 2-fold increase in AQP5 correlated with increased effusion viscosity (1.94 mL/mg; 95% CI, 0.08-3.80 mL/mg). Conclusions and Relevance:Further exploration of the role of MUC5B in the pathophysiology of OME holds promise for development of novel, targeted therapies to reduce effusion viscosity, facilitation of effusion clearance, and prevention of disease chronicity and hearing loss in patients with OME.

Dataset Information

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains.

Publications

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains.

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