Calcium-dependent protein interactions in MUC5B provide reversible cross-links in salivary mucus

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 concentration-dependent 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 x 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 CaCl2 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 x 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.