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 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 106) 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 Kd 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.
Highlights
Mucus forms a viscoelastic gel that coats the epithelial surfaces in humans and other vertebrates
The results showed that purified MUC5B formed concentrated solutions in physiological saline in which there was no evidence of self-association, and the properties at high concentration were as predicted by molecular entanglement of the oligomeric mucins [15]
Comparison of native saliva with the solutions of guanidinium chloride (GdmCl) purified MUC5B under similar ionic conditions revealed that saliva had much lower porosity than the purified mucin at comparable concentrations, showing that saliva contained an additional level of organization that was absent from the purified MUC5B mucin [15]
Summary
Materials—Trypsin, modified by reductive alkylation to reduce autolysis, was purchased from Promega (Southampton, UK). MUC5B mucin fractions, in the void volume of the column, were pooled and dialyzed against 0.1 M NaCl, pH 6.5, and part was concentrated against polyethylene glycol (5% w/v) and tested for calciumdependent effects on tracer diffusion by confocal-FRAP. A 5-ml aliquot of the pooled high density MUC5B mucin-containing fraction was chromatographed on a size exclusion column (600 ϫ 10 mm, Sepharose CL-2B) eluted in 0.1 M NaCl, pH 6.5, containing 10 mM CaCl2 at a flow rate of 60 ml/h. Calcium binding was determined (as above) after treatment of the high density mucin fraction with 6 M GdmCl. A sample of the mucin was dialyzed into 6 M GdmCl (pH 6.2) overnight at 4 °C and back into 0.1 M NaCl, pH 6.5, prior to addition of radioactive 45CaCl2. The samples were chromatographed on PD-10 size exclusion columns (Sephadex G-25) eluted in 25 mM NaCl, 20 mM Tris, pH 7.0, and the radioactivity in fractions (500 l) was measured by scintillation counting
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