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 × 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 K d of 7 × 10–6m, 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. 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 × 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 K d of 7 × 10–6m, 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 (1Carlstedt I. Sheehan J.K. Corfield A.P. Gallagher J.T. Essays Biochem. 1985; 20: 40-76PubMed Google Scholar, 2Verdugo P. Aitken M. Langley L. Villalon M.J. Biorheology. 1987; 24: 625-633Crossref PubMed Scopus (69) Google Scholar). Mucus has also been described as a network weakly cross-linked by non-covalent bonds (3Crowther R.S. Marriott C. James S.L. Biorheology. 1984; 21: 253-263Crossref PubMed Scopus (41) Google Scholar, 4Steiner C.A. Litt M. Nossal R. Biorheology. 1984; 21: 235-252Crossref PubMed Scopus (20) Google Scholar) and suggested mechanisms of interaction have included interchain hydrophobic interactions (5Bromberg L.E. Barr D.P. Biomacromolecules. 2000; 1: 325-334Crossref PubMed Scopus (62) Google Scholar) and carbohydrate-carbohydrate interactions between the mucins (6McCullagh C.M. Jamieson A.M. Blackwell J. Gupta R. Biopolymers. 1995; 35: 149-159Crossref PubMed Scopus (36) Google Scholar, 7Sellers L.A. Allen A. Morris E.R. Ross-Murphy S.B. Biochim. Biophys. Acta. 1991; 1115: 174-179Crossref PubMed Scopus (38) Google Scholar, 8Shogren R. Jamieson A.M. Blackwell J. Cheng P.W. Dearborn D.G. Boat T.F. Biopolymers. 1983; 22: 1657-1675Crossref PubMed Scopus (26) Google Scholar, 9Soby L.M. Jamieson A.M. Blackwell J. Jentoft N. Biopolymers. 1990; 29: 1359-1366Crossref PubMed Scopus (15) Google Scholar). Mucus rheology is affected by many factors, including hydration (2Verdugo P. Aitken M. Langley L. Villalon M.J. Biorheology. 1987; 24: 625-633Crossref PubMed Scopus (69) Google Scholar), pH (10Verdugo P. Annu. Rev. Physiol. 1990; 52: 157-176Crossref PubMed Scopus (243) Google Scholar), and ion content (11Glantz P.O. Wirth S.M. Baier R.E. Wirth J.E. Acta Odontol. Scand. 1989; 47: 7-15Crossref PubMed Scopus (14) Google Scholar, 12Saltzman W.M. Radomsky M.L. Whaley K.J. Cone R.A. Biophys. J. 1994; 66: 508-515Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 13Beeley J.A. Biochem. Soc. Trans. 1993; 21: 133-138Crossref PubMed Scopus (31) Google Scholar). 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 (14Madsen F. Eberth K. Smart J.D. J. Control Release. 1998; 50: 167-178Crossref PubMed Scopus (176) Google Scholar). 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 the attendant problems of poor gas exchange, bacterial colonization, and inflammation (10Verdugo P. Annu. Rev. Physiol. 1990; 52: 157-176Crossref PubMed Scopus (243) Google Scholar). To gain further insight into mucus organization we have recently employed diffusion analysis by fluorescence recovery after photobleaching using a confocal microscope (confocal-FRAP) 1The abbreviations used are: confocal-FRAP, fluorescence recovery after photobleaching using a confocal microscope; GdmCl, guanidinium chloride; DTT, dithiothreitol; ESI-MS-MS, positive ion electrospray ionization-tandem mass spectrometry; MALLS, multiangle laser light-scattering. to study the properties of saliva (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). This technique provides a powerful approach to investigate the properties of macromolecules in concentrated solution (16Gribbon P. Hardingham T.E. Biophys. J. 1998; 75: 1032-1039Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). It enables molecular mobility to be determined in complex mixtures in the absence of flow and shear forces. In initial work we characterized the concentration dependence of the self-diffusion of the MUC5B mucin, the predominant oligomeric mucin present in saliva. The measurement of lateral translational diffusion of fluorescent microspheres of different size in solutions of purified MUC5B mucin and in native saliva was used to provide a direct estimate of the porosity (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar, 17Gribbon P. Hardingham T.E. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar, P. Hardingham T.E. Biochem. J. 2000; PubMed Scopus Google Scholar, A. J. M. 1994; Scopus Google Scholar, J. 1989; Scopus Google Scholar). The results showed that purified MUC5B concentrated solutions in physiological in was of and the properties at high concentration as by molecular entanglement of the oligomeric mucins (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). hydrophobic interactions between mucins carbohydrate-carbohydrate interactions (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). of native saliva with the solutions of guanidinium chloride purified MUC5B that saliva much porosity the purified mucin at that saliva contained an of organization that was from the purified MUC5B mucin (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). In the present we investigated the basis of the organization present in reduced tracer by to was from was from Calcium and chloride from chloride and from solutions of guanidinium chloride with The was from and Sepharose and size from was from and chloride was from and of MUC5B saliva was from and at × for at to The was with a pH of It was at and was used within and showed change in pH or properties this MUC5B mucin concentration was determined by measurements as described (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). and of saliva m mm pH the with 10 mm or the with 10 mm The of was as For molecular weight the on a × 10 mm) with at a flow of The mass average molecular mass was determined using an multiangle laser and a For analysis of the weight average molecular weight was by the major high molecular weight mucin between 6 and of with a of in an at a of in to concentration between and For the flow in the was and and and flow and is the MUC5B mucin The intrinsic viscosity was by the reduced viscosity as a of mucin concentration the line at concentration gave the intrinsic line MUC5B from mucins purified from by (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). was in 6 m and by size and in m This was into solutions to from saliva was at with an of m NaCl. The pH was × the was by in m pH in a at for at The high density MUC5B mucin against m pH and at after the addition of A fraction of this was on a size × Sepharose in m 10 mm pH at a flow of MUC5B mucin in the of the and against m pH and was concentrated against and for effects on tracer diffusion by by of the high density MUC5B fraction purified by density was reduced (10 mm for 2 and mm for in the and against and In a of the high density MUC5B fraction was on a size × 10 Sepharose in m pH 10 mm CaCl2 at a flow of The fraction was reduced and as described at with of 10 of protein in mm pH by and by positive ion using a mass a with a of the on a × mm) with a of in to in at a flow of This was for and in the first excluded from the analysis. using and using the to mass and The of was with the being that the and ion within the and that the contained a of at of for tracer diffusion fluorescent polystyrene microspheres with (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar), with the and at measurements on saliva and MUC5B solutions in the presence of m pH and at in the on dependence in for at from For the concentration dependence of tracer saliva was concentrated by against For measurements in the presence or absence of 10 mm the concentration of MUC5B in the saliva was up to In the presence of 10 mm EGTA the concentration of MUC5B was up to MUC5B purified in m pH and measurements on solutions up to To the effects of on tracer diffusion the of and or EGTA to up to To the of the after the addition of EGTA was to a concentration to the CaCl2 and the measurements To the effect of on tracer up to was to saliva by recovery after photobleaching using a confocal laser microscope with an microscope (16Gribbon P. Hardingham T.E. Biophys. J. 1998; 75: 1032-1039Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). For tracer diffusion microspheres on a microscope The technique the lateral translational diffusion from the dependence of the of the of the of (16Gribbon P. Hardingham T.E. Biophys. J. 1998; 75: 1032-1039Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Gribbon P. Hardingham T.E. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar, P. Hardingham T.E. Biochem. J. 2000; PubMed Scopus Google Scholar, P. R. Biophys. J. 1994; Full Text PDF PubMed Scopus Google Scholar). for In the dependence the was using a and to the on the confocal The concentration dependence of translational diffusion of the tracer in saliva and purified MUC5B was using the for tracer diffusion in J. 1989; Scopus Google Scholar) in is the apparent lateral translational tracer diffusion is free diffusion and and and to the average pore size of the network A. J. M. 1994; Scopus Google Scholar) the between entanglement in Scholar) in In this d is the of the This that the forms a network of For as mucin in interchain and the network be is an average a of apparent pore In solutions of this analysis of tracer an apparent pore provides a of with effect of specific interchain Calcium binding to the high density MUC5B mucin fraction purified mild was by size To the mucin fraction at in m pH was to a concentration of and at The was on a of Sepharose × mm) in m pH and the was by Calcium binding was also determined after treatment of the high density mucin fraction with 6 m A of the mucin was into 6 m at and into m pH to addition of The high density mucin fraction was also by 10 mm for at to with calcium at and To estimate the of calcium binding different of to to of the high density mucin fraction concentration in m pH The on size in mm mm pH and the in was by work tracer diffusion of fluorescent polystyrene microspheres by showed that MUC5B mucin was much to tracer diffusion saliva at of MUC5B mucin (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). investigated the properties of saliva further to the basis for this in molecular diffusion in saliva and MUC5B solution showed a concentration-dependent reduction in diffusion of microspheres This the for entanglement of high molecular weight J. 1989; Scopus Google Scholar). the results of tracer diffusion the change in apparent pore size with concentration was This showed that the apparent pore size in MUC5B solution was larger in to MUC5B concentration to that The was due to both in m NaCl. To the basis for the in apparent pore we further tracer diffusion on saliva at of MUC5B in the of the this concentration change in tracer diffusion in the organization of the macromolecules in saliva that the of of Calcium and EGTA on addition of up to mm to saliva in m caused a major in the diffusion of the from × to × these diffusion the apparent pore size in saliva from from to nm after addition of mm CaCl2 In a at the saliva EGTA tracer diffusion and at mm EGTA the diffusion to the free The suggested that EGTA was calcium in saliva and an increase in tracer The apparent pore size from to nm in the presence of mm EGTA The apparent pore size of saliva from tracer diffusion measurements was by These major in tracer diffusion at a concentration of the saliva suggested that calcium has a major effect on the organization of the macromolecules in saliva. The of this effect was shown by analysis of a with EGTA and with calcium and The tracer diffusion by EGTA to a in the presence of calcium The calcium effect was of pH in the pH The suggested that calcium was and from of interaction and that the change in macromolecular organization in saliva was A of the effects of calcium on tracer diffusion the of saliva concentration was There was a major reduction of tracer diffusion the concentration with with the effect at of MUC5B In in the presence of the saliva was much to tracer and effects on microsphere diffusion these results the apparent pore size showed a 20-fold between and saliva at a physiological the tracer diffusion of the microspheres in MUC5B mucin solution showed a concentration dependence as in saliva with EGTA of tracer diffusion × size at of 10 mm 10 mm MUC5B 10 mm in a These initial of calcium effects on tracer diffusion investigated further by other The viscosity of saliva was to that determined by R.S. J. 1984; Scholar, M. J. 1989; Google Scholar). with the addition of 10 mm was a increase in intrinsic viscosity to and with 10 mm EGTA was a in intrinsic viscosity to ml/g) and The presence of calcium caused the saliva to be Analysis of native saliva by gel on with analysis showed it to contain a high molecular weight fraction with an approximate weight average molecular weight of × In the presence of CaCl2 (10 mm) the molecular weight of the high molecular weight fraction showed a major increase to × and this was reduced to × in the presence of EGTA (10 mm) and Calcium caused a major increase in the viscosity of saliva and the average molecular weight of the high molecular weight in and molecular weight average molecular 10 mm 10 mm in a of and and on in effect of calcium on tracer diffusion in saliva was unaffected by concentration to 2 m The calcium effect was to be due to interaction the macromolecules within saliva. The effects of other addition of and up to mm was change in tracer diffusion The results showed that other for calcium and that calcium has a specific effect on macromolecular organization in saliva. of on in diffusion was determined on saliva at different the the tracer diffusion of the microspheres by the of for the effect of viscosity of the on tracer The effect was on the diffusion of the microspheres and to the initial these was an increase in the apparent pore size with from nm at to nm at These results suggested that the porosity of saliva to tracer diffusion was on a of calcium binding that was affected by with at with of on in investigate the of the interactions that saliva we investigated the effect of the tracer diffusion was by the presence of up to This gave that be with the mucin, in the effect on saliva. Calcium to for calcium binding to the macromolecules in of the saliva was by density mild to the high density MUC5B mucin from other ion analysis of from this high density fraction that it was in MUC5B also contained of and of this fraction on Sepharose after with showed was calcium binding to a high molecular weight fraction in the of the This showed that the calcium binding with the MUC5B The high density MUC5B mucin fraction was also purified further by size on Sepharose in the presence of 10 mm of the different size of and Sepharose the major high molecular weight fraction in the presence of EGTA to the of the and this fraction the effects on tracer It showed a in tracer diffusion of in the presence of 10 mm CaCl2 × to showed that the effect was on a molecular weight protein by The high molecular weight MUC5B fraction from the after Sepharose in the presence of CaCl2 (10 mm) was after by positive ion The results showed that it was purified and the present in the high density mucin MUC5B was the protein and of the MUC5B and was from a MUC5B has a high molecular this technique other present at These results that the calcium binding is a of MUC5B by in MUC5B in a of the high density mucin fraction with 6 m by in m to Sepharose in of calcium binding This suggested that used MUC5B mucin caused the of of tracer diffusion in mucin that was effect of calcium A and Calcium binding was also from the MUC5B fraction after mild reduction with at The of calcium binding to a of the high density MUC5B mucin fraction was determined by with different of to and on analysis of the calcium at different gave an estimate of the of calcium binding to the mucin fraction of × The approximate concentration of MUC5B in this fraction was 7 × × as the MUC5B mucin molecular weight (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google and from this concentration the of calcium binding was to be binding of MUC5B These results suggested that MUC5B contained a protein that was for calcium binding and was sensitive to mild reduction and to by we showed by measurements of tracer diffusion that concentrated solutions of MUC5B the properties of saliva (15Raynal B.D. Hardingham T.E. Thornton D.J. Sheehan J.K. Biochem. J. 2002; 362: 289-296Crossref PubMed Scopus (70) Google Scholar). It was that an in the organization of the mucins in and we present that interactions for this in Calcium a major effect on the of saliva to tracer diffusion a 20-fold change in the apparent pore size of saliva from a to a The effects of calcium on the tracer diffusion in saliva after treatment by EGTA and specific to calcium. the addition of calcium to saliva in the of a high molecular weight macromolecular fraction with a increase in weight average molecular weight and a increase in intrinsic The calcium-mediated effect on tracer diffusion in saliva was at from m up to m NaCl. This the present results from (3Crowther R.S. Marriott C. James S.L. Biorheology. 1984; 21: 253-263Crossref PubMed Scopus (41) Google Scholar, J.A. J.T. 1989; Full Text PDF PubMed Scopus Google Scholar) effects on mucin viscosity and rheological properties with concentrations. These have from interactions between mucins at used in this The results in also showed for interactions due to as has been with high concentration of purified mucin Blackwell J. Jamieson A.M. M. Biorheology. PubMed Google Scholar). The of the calcium effect on the saliva properties suggested it to be by an on free calcium of calcium in saliva by EGTA porosity to tracer diffusion to to that of MUC5B this also caused a in the intrinsic viscosity (733 with ml/g) and calcium ml/g) saliva. that MUC5B from saliva calcium effect on tracer This to be due to the of calcium treatment with 6 m destroyed the calcium binding of MUC5B mucins in the absence of treatment calcium it for the between the intrinsic viscosity of mucins J.K. I. Biochem. J. 1984; PubMed Scopus (69) Google Scholar), have also been shown to be MUC5B C. I. Biochem. 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Sheehan J.K. PubMed Scopus Google Scholar) have used 6 m as a to the of results that treatment the calcium effects in MUC5B as the this in the effects on saliva. The present results that calcium binding to protein in MUC5B mucin the of MUC5B into larger This is an organization in addition to the characterized oligomeric of There in the MUC5B protein including to and a in other have been shown to calcium D.P. Biochem. Biophys. 1998; PubMed Scopus Google Scholar). The present results that the saliva properties by interactions physiological the concentration of calcium in saliva is between and 2 the of calcium concentration provides a that physical This calcium-mediated to other of mucus the MUC5B mucin, as respiratory and and for and the for