Calcium-dependent Protein Interactions in MUC5B Provide Reversible Cross-links in Salivary Mucus

Abstract

The macromolecular organization within saliva was investigated by tracer diffusion measurements of fluorescent polystyrene microspheres by fluorescence recovery after photobleaching using a confocal microscope (confocal-FRAP). There was a concentrationdependent reduction in microsphere diffusion; this was much greater in the presence of calcium (10 mM) and was reduced by the addition of EGTA (10 mM). These effects on tracer diffusion showed that native saliva contained a macromolecular organization that was sensitive to free calcium concentrations. This was supported by a major increase in the weight average molecular weight of the high molecular weight mucin fraction in saliva (10 -62 ؋ 10 6 ) and an increase in intrinsic viscosity of saliva (733 to 1203 ml/g) both caused by calcium. Analysis of the change in tracer diffusion in saliva showed a 20-fold increase in the apparent pore size (from 130 nm in 10 mM CaCl 2 to 2600 nm in 10 mM EGTA at physiological concentration). The effect was specific for calcium and was unaffected by up to 2 M NaCl. The calcium binding activity was contained in a high buoyant density fraction of saliva excluded from Sepharose CL-2B. Calcium binding to this fraction gave an approximate K d of 7 ؋ 10 ؊6 M, and the binding was irreversibly destroyed by treatment with 6 M guanidinium chloride and by mild reduction, suggesting it to be to a protein site. This fraction of saliva was shown to contain MUC5B as the single major protein species by positive ion electrospray ionization-tandem mass spectrometry analysis. The results suggested that oligomeric MUC5B in saliva is assembled into much larger linear or branched assemblies through calcium-mediated protein cross-links.Mucus forms a viscoelastic gel that coats the epithelial surfaces in humans and other vertebrates. The properties of the gel have been interpreted as being predominantly due to entanglement of the long, high molecular weight oligomeric mucins (1, 2). Mucus has also been described as a network weakly cross-linked by non-covalent bonds (3, 4) and suggested mechanisms of interaction have included interchain hydrophobic interactions (5) and carbohydrate-carbohydrate interactions between the mucins (6 -9). Mucus rheology is affected by many factors, including hydration (2), pH (10), and ion content (11-13). Thus the supramolecular organization of the mucus layer is complex, and the molecular basis of this organization remains poorly defined.A layer of mucus coats the surfaces of the gastrointestinal, reproductive, and respiratory tracts as well as the eyes and oral cavity (14). This layer is the bodies' first line of defense against chemical, physical, and biological insult and a change in this barrier will compromise health. For example, in the airways overproduction of mucus with aberrant rheological properties is a feature of asthma, cystic fibrosis, and chronic obstructive pulmonary disease. In these situations the change in the physical properties of the barrier leads to a breakdown in mucus clearance from the airways with...

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