1. Mucin Structure The Structure and Heterogeneity of Respiratory Mucus Glycoproteins 1 - 3 JOHN K. SHEEHAN, DAVID J. THORNTON, MARGARET SOMERVILLE, and INGEMAR CARLSTEDT
Introduction In the normal respiratory tract, inhaled matter such as dust, microorganisms, and chemical irritants is removed mainly by mucociliary transport. The coordinated beating of cilia propels an overlying layer of mucus toward the pharynx, where it is swallowed (1). In health, the amount of respiratory secretions reaching the trachea is very small (2); however, far greater amounts may be produced in conditions such as chronic bronchitis, asthma, and bronchiectasis. Airway mucus is a mixture of water, salts, protein, and high M, glycoconjugates. Mucus glycoproteins(mucins) are accepted as major constituents of mucus in hypersecretory conditions, but there is disagreement about the macromolecular composition of normal respiratory secretions. Bhaskar and coworkers (3, 4) have proposed that the main glycoconjugate from unstimulated healthy airways is proteoglycan, whereas we have observed mucus glycoprotein as the maj or secretory product and little if any proteoglycan (5). The mucus glycoproteinsconfer on mucus its characteristic properties of viscosity and elasticity, which enable it to be transported by cilia. Several cell types in the respiratory mucosa produce mucus glycoproteins, including the mucous cells in the submucosal glands and the goblet, and perhaps the ciliated, cells in the surface epithelium (6, 7). The control of respiratory mucus secretion is complex. The submucosal glands receive a dense innervation from the autonomic nervous system (8, 9), and both cholinergic and adrenergic agonists stimulate macromolecular output (10). In addition, neuropeptides (11), inflammatory mediators (12, 13),bacterial products (14, 15), and irritants (16, 17) have all been shown to modify mucus output. Histochemical studies (18, 19)have indicated the presence of different types of mucins, i.e., neutral and acidic (sulfated or sialylated), and these observations, as well as those made by using lectin histochemistry (20),suggest that different cells may produce structurally distinct mucins. Thus, respiratory mucus may contain a number of distinct populations of mucus glycoproteins and this, together with the complex control of secretion, presents scope for fine tuning of the response to a particular stimulus. However, there is as yet no biochemical evidence for different cellssynthesizing different mucins. The macromolecular structure of respiratory mucus glycoproteins has been the subject of some debate, and the recent literature indicates that opinions still differ as to the 54
SUMMARY Respiratory mucus glycoprotelns purified from both "normal" respiratory secretions and sputa of patients with a variety of hYPersecretoryconditions are high M, linear molecules adopting a random coil configuration in solution. Studies on their polydisperslty show them to have an M, In the range 3 to 32 X 106 and a distribution of length from 200 nm to beyond 10 J.1m. These macromolecules are fragmented by reduction of intermolecular disulfide bonds into subunits, with M, approximately 2 x 106 and length from 200 to 600 nm. Reduction not only cleaves the mucin molecule but opens, presumably by breaking Intramolecular disulfide bonds, cryptic "naked" protein regions. Trypsin digestion of subunits yields high M, glycopeptldes (M" 300 to 500,000), presumably by cleavage of the peptide core within the unfolded "naked" protein domains. Respiratory mucus glycoprotelns from infected sputum samples are usually heterogeneous In CsCI density gradients, In contrast to those from "normal" tracheobronchial secretions. The former are characterized by the presence of a number of different mucin species, and the basis for the separation of these mucins appears to be the variable presence of sialic acid and sulfate moieties In the oligosaccharide clusters. This heterogeneity may reflect a difference In cellular origin of the mucins and also may be clinically significant. AM REV RE8PIR DI8 1991; 144:84-89
size and heterogeneity of whole mucins as well as the existenceof a subunit structure for these molecules (21-24). However, it is agreed that proteolysis of these macromolecules yields a glycopeptide of M, 300 to 500,000, with a protein core rich in proline, serine, and threonine, which is densely substituted by a diverse range of O-linked oligosaccharides. These oligosaccharides have been the subject of detailed structural studies (25-29), but as to whether they are attached to distinct proteins or whether they have a particular organization along the protein core is not known.
Isolation/Purification In order to study the macromolecular properties of mucus glycoproteins, it is important to maintain, as far as possible, the integrity of the core protein throughout the isolation and purification process. Thus, high shear extraction methods and covalent bond-breaking reagents (i,e., dithiothreitol) should be avoided, and measures should be taken to suppress endogenous proteolytic activity in the samples. To this end we have adopted the procedure devised for the isolation and purification of cervical mucins (30, 31).In brief, the mucous gel is extracted with 6M-guanidinium chloride (GuHCI) in the presence of protease inhibitors. Insoluble material is removed by centrifugation, and the extractant is brought to 4M-GuHCI before the addition of CsCI to a final density of approximately 1.4g/ml. In our experience the gradient forming salt (typically CsCI) should not be added to the sample during extraction since the mucous gel may not be fully dispersed, and conventional high-speed centrifugation cannot then be employed to remove nonsolubilized material. Nonmucin proteins are removed by isopycnic centrifugation; two rounds are usu-
ally necessary for their complete removal, and a further purification step in CsCI/0.2MGuHCI removes contaminating DNA. This procedure works well even with heavily infected sputa in which large amounts of nonmucin protein and DNA are present (32). When working with sputum, enough mucin can be prepared from a single subject to permit a full characterization. So little mucus is produced by the healthy respiratory tract, however, that the main problem in studying these secretions lies in obtaining them in sufficient amounts from a single subject, and it is therefore necessary to pool secretions from a number of subjects (5).
Criteria for Purity We assay our samples by polyacrylamide gel electrophoresis with subsequent silver staining to reveal protein contamination and by analytical ultracentrifugation using A 2so monitoring for DNA. The specificabsorbance 1 From the Department of Biochemistry & Molecular Biology, University of Manchester, Manchester, United Kingdom, and the Department of Physiological Chemistry 2, University of Lund, Lund, Sweden. 2 Supported by the WellcomeTrust, UK; the Cystic Fibrosis Research Trust, UK; the Medical Research Council, UK; the British Lung Foundation; Grant No. 7902 from the SwedishMedical Research Council; the Swedish National Association against Heart and Chest Diseases; the National Swedish Environmental Protection Board; the Swedish Society for Cystic Fibrosis; Greta och Johan Kocks Stiftelser, Trelleborg, Sweden; the Medical Faculty, University of Lund, Sweden; and the National Swedish Board for Technical Development. 3 Correspondence and requests for reprints should be addressed to John K. Sheehan, Department of Biochemistry & Molecular Biology,University of Manchester, Manchester M13 9PT, UK.
55
STRUCTURE AND HETEROGENEITY OF MUCINS
for DNA is approximately 100 times that for mucins, making this a particularly sensitive assay. We estimate that contamination by nonmucin protein and DNA is in general below 1070 (wt/wt). It has been proposed that mucins contain a significant amount of lipid and also covalently bound fatty acids (33-35). However, when we prepared chronic bronchitic mucins by the procedure outlined above and extracted with organic solvents only small amounts of fatty acids (0.9 ug/rng of mucin) were detected, and no glycolipids, phospholipids, cholesterol, or triglycerides could be found (36). After lipid extraction, the mucins were subjected to alkaline methanolysis to release any covalently bound fatty acids, and the amount detected ranged from zero to 25 ng/mg of glycoprotein, Le., at the limits of detectability. We conclude that there are no detectable fatty acids covalently bound to respiratory or other mucins, though there are hydrophobic domains on these macromolecules (37), which may be responsible for the tight binding of fatty acids.
Size and Shape The size of respiratory mucus glycoproteins has been investigated by a number of workers, and a broad range of M, values have been reported (table 1). We have studied respiratory mucins from healthy human airways and from patients with asthma, cystic fibrosis (CF), or chronic bronchitis (5, 32). Values of M, were determined by using absolute-intensity, light-scattering methods (Zimm Plot analysis) and with sedimentation and diffusion measurements. A typical Zimm plot for a chronic bronchitic mucin is shown in figure 1 and yields values of M, and the radius of gyration (Rn) of 21 x 106 and 290 nm, respectively. Analytical ultracentrifugation indicates that the material is homogeneous although polydisperse in size with a marked concentrationdependent sedimentation rate (figure 2A). Quasi-elastic light-scattering measurements were performed to obtain the translational diffusion coefficient (Di), and extrapolation to both zero angle and concentration is necessary to obtain accurate values (figure 2B). We have obtained values of weight-average M, ranging from 14 to 25 x 106 for mucins isolated from both pooled "normal" respiratory secretions and chronic bronchitic sputum TABLE 1 SIZE OF RESPIRATORY MUCINS
Mr (x 10-6 )
Reference No.
3.5 1.5 2.3
22 23 38
CF sputum
9.3 17
21 32
Chronic bronchitic sputum
5-7 1.0
39 40
Source Asthmatic sputum
Normal secretions Definition of abbreviation: CF
14
= cystic
5 fibrosis.
10
Fig. 1. Zimm plot of light-scattering data for chronic bronchitic mucins. Readings were taken at 9 = 20,30,40,50, 60,75,90,110, and 130 degrees (closed circles) and extrapolation to 9 = 0 (open circles) was performed from 20 to 40 degrees. The concentrations used were 0.079,0.158,0.237, and 0.316mg/ml and extrapolation to zero concentration (open circles) yielded the radius of gyration. The optical constant was 9.32 x 10-8 cm 2g-2 •
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(eight different subjects). In contrast, mucins isolated from CF sputum are of lower M, (32, 41); however, after gel-chromatography on Sepharose CL-2B the molecules excluded from the gel are similar in size to those found in normal secretions and chronic bronchitic sputa (32). In general, the values of M, obtained by us are higher than those reported by others (see table 1). This discrepancy may be accounted for by different preparation methods and sample handling. The values of Mr discussed above are averages for the whole distribution of molecules and tell us little about their range of mass and size.Equilibrium centrifugation (42), ratezonal centrifugation in GuHCI gradients (43), and gel chromatography on Sephacryl S-loo0 (21) are methods that can be used to investigate the polydispersity of macromolecules. A typical rate-zonal profile for CF respiratory mucins is shown in figure 3A. It is unlikely that the observed polydispersity is due to weak aggregation effects because fractions from the gradient (10 to 20 times more dilute) re-run close to their original position (figure 3B). Absolute-intensity, light-scattering measurements on fractions taken across the gradient yield values of M, in the range of 3 to 32 X 106 and Rn in the range of 100 to 490 nm.
The relationship between Ro and M, can be expressed as a Mark-Houwinck-type equation: Ro = kMa, where a is a shape-dependent parameter (44). A plot of log Ro against log M, (figure 3C) yields a line of slope a = 0.66, which is consistent with the mucins being linear and flexible molecules behaving as expanded random coils in solution. Similar findings have been reported for chronic bronchitic, pig gastric (45), CF respiratory (21), human cervical (44), and pig submaxillary (46) mucins. The architecture of respiratory mucins has been examined by a number of investigators using electron microscopy (47-53). There is agreement between these studies in that the molecules appear as flexible random coils, polydisperse in size, with lengths from 0.2 to 5 urn, However, the length distributions of the mucins vary between the different groups, presumably reflecting differences in sample purification and preparation for electron microscopy. Harding and coworkers (48), studying CF mucins and using a critical drying procedure, visualized the molecules as "beaded" structures with distinct globular domains occurring regularly along a filamentous thread. Rotary shadowing methods also have been used (49-51, 53). In these studies the mole-
56
SHEEHAN, THORNTON, SOMERVILLE, AND CARLSTEDT
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Fig. 3. Polydispersity of CF mucins. In panel a, mucins (closed circles) were fractionated by using rate-zonal centrifugation on a guanidinium chloride gradient (described in references 5 and 43). The fractions were analyzed with the periodic acid-Schiff reaction after slotblotting onto a nitrocellulose membrane, and the color yield (integrated intensity) was quantitated with an image analysis system used as a reflectance densitometer (62). The concentration of guanidinium chloride (dashed line) was determined by refractive index measurements. In panel b, fractions 3 (closed circles), 8 (open circles), and 11(closed squares) from (a) were resubjected to rate-zonal centrifugation. In panel c, values of M r and RG across the mucin distribution of (a) were determined from absolute-intensity light-scattering data and are plotted as log-RG against log-Mr.
8.0
Fig. 4. Electron microscopy of "normal" respiratory mucins. The. macromolecules were spread in benzyldimethylalkylammonium chloride monolayers on a hypophase of (a) 6M-GuHCI and (b and c) 0.2 M NaCI, picked up on carboncoated grids, and made visible by rotary shadowing with platinum/tungsten (52).
culesweresprayed onto mica, and subsequently a replica was formed by shadowing with platinum and carbon. This technique, though applicable to most proteins, is not in general suitable for very large flexible molecules (i.e., mucus glycoproteins and DNA) where the high shear forces involved in atomizing the droplets may fragment such structures. For example, even triple-helical collagen molecules are sheared by this procedure (54). We have studied respiratory mucins using spreading techniques as employed in visualizing DNA (55, 56). The advantages of this approach are 2-fold. First, it avoids high shear forces during deposition of the molecules on the grid, and second, it is possible to deposit molecules in the presence of the same denaturing solvent (6M-GuHCI) that was used for characterizing the molecules in solution. The appearance of "normal" respiratory mucins, spread in 6M-GuHCI, is shown in figure 4A. The moleculesare convoluted,flexiblethreads, polydisperse in length, ranging from 300 nm to several microns, although their precise lengths are difficult to measure because of their tortuous nature and entanglement. The molecules are similar after spreading in 0.2 M NaCI, but they are less convoluted (figure 4B). In all solvent systems employed, there is evidence of heterogeneity, with a proportion (approximately 10070) of the molecules having a distinctly lower contrast (Le., thinner) than the rest (figure 4B). Mucins with a randomly meandering configuration typically have an uneven and "beaded" appearance, whereas those distorted under flow appear as smooth and thin filaments, often more than 10 urn in length (figure 4C). Thus, it appears that mucins have regions that are highly folded and condensed and that can be "unfolded" under the influence of mechanical forces. If true, this could have important consequences for the rheologic properties of a mucous gel where elastic energy may be stored within individual molecules as wellas the network of the gel.
Fragmentation of the Protein Core We and others (5, 21, 32, 39) find distinct changes in molecular size of respiratory mucins after reduction. In studies on "normal" human (5) and CF respiratory mucins (32), employing gel chromatography and electron microscopy, we have shown that the major fragment obtained after reduction of disulfide bonds is significantly smaller than whole mucins and is visualized as a linear flexible filament 200 to 600 nm in length. The chromatographic behavior of this fragment is similar to the reduced CF respiratory mucins isolated by Rose and coworkers (57), and it is thus very similar to subunits (M, 2 x 106 ) prepared from cervical mucins (58). Therefore, we propose that respiratory mucins are composed of subunits similar to those from cervical mucins. This is in agreement with the data of Gupta and coworkers (21) for CF respiratory mucins, but the existence of this subunit structure is not accepted by all workers in the field. For example, Chace and co-
57
STRUCTURE AND HETEROGENEITY OF MUCINS
workers (22), using light-scattering techniques, observed no decrease in M, (3.5 to 3.8 x 106 ) of asthmatic and CF respiratory mucins after reduction, whereas Roussel and coworkers (24) have suggested that the effects of reducing agents may be explained without involving the rupture of intermolecular disulfide bonds. Subunits, prepared from chronic bronchitic, gastric, and salivary mucins (59) as well as CF mucins (unpublished observation), crossreact with polyclonal antibodies directed towards "cryptic" protein domains exposed after reduction of whole cervicalmucins (59). These data indicate that respiratory mucins share protein epitopes with human cervical, gastric, and salivary mucins. Proteolysis of mucus glycoproteins releases a major population of large glycopeptides and smaller peptide and glycopeptide fragments, the latter probably originating from the "naked" protein regions on the core. The value of M, of the major glycopeptide fragment is dependent upon the protease employed (e.g., papain generates smaller molecules than does trypsin) and the degree of substitution and size of the oligosaccharides. Trypsin digestion of subunits generates smaller fragments than those obtained by direct digestion of mucins, and we distinguish between these situations by calling the former "T-domains" and the latter "tryptic glycopeptides." The high M, glycopeptides correspond to the dense "oligosaccharide clusters" found periodically along the molecule, and our studies on these structures indicate that they have a M, of 300 to 500,000, with lengths between 80 and 180 nm. A schematic diagram of the proposed mucin structure is shown in figure 5.
Heterogeneity Isopycnic centrifugation employed for the purification of respiratory mucins suggested to us the presence of different macromolecular species in many samples. Whereas "normal" mucins appeared as a homogeneous, almost Gaussian, distribution in the CsCI/ 0.2M-GuHCI density gradient, those from patients with CF (32) or other hypersecretory conditions (Somervilleand coworkers, unpublished observations) wereoften heterogeneous (figure 6). The resolution in an isopycnic density gradient is dependent on the M, of the molecules, the band width being related to the M, according to the relationship (60): 0 2 = RT PolM r(jj 2 rs (dp/dr), where Po is the initial solution density, 0 is the Y2 band width at 0.607 x peak height, co is the angular velocity of the rotor, rs is the distance from the rotor center to the band peak, and dp/dr is the density gradient at the band peak. Polydispersity of M, and nonideality effects caused by high concentration of the mucins, often a feature of preparative work, broaden density distribution profiles and obscure heterogeneity. To overcome this we have fractionated, by using rate-zonal centrifugation on GuHCI gradients, purified bronchiectatic mucins in order to obtain molecules (about 20070 of the total) with molecular
(a)
/~
Fig. 5. Schematic diagram of the proposed "architecture" of respiratory mucins. In panel (a), the whole mucins are made up of subunits joined end to end by disulfide bonds (s-s). (b) Upon reduction, subunits are released that contain alternatingoligosaccharideclustersand "naked" protein domains. Immunologic data suggest that the latter become unfolded by reduction (59). Trypsindigestion of subunits yields high Mr glycopeptides (T-domains) corresponding to the oligosaccharide clusters. These clustersare represented as "rodlike" only for the convenience of drawing. SU = freethiol generatedafter reduction of s-s.
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'naked' stretch of protein weights in excess of 15 x 106 • When resubjected to density-gradient centrifugation in CsCI/0.2 M-GuHCI the mucins band more sharply, and the heterogeneity is more pronounced (figure 7A). The data confirm that the molecules are of high M«, which, if because of self-association, implies that aggregates must be very stable and specific, with the molecules not disposed to heterologous interactions. Although mucins may be fractionated into different species using this approach, it is impractical because of the small proportion of the mucin population utilized and the dilution required to achieve the fractionation. The observations on heterogeneity at the whole mucin level predict that the macromolecules are constructed from different types of "oligosaccharide clusters." These structures, which are a feature of all respiratory mucins we have studied, are smaller, more homogeneous in size, and thus more amenable to fractionation by high-resolution techniques
such as ion-exchange high performance liquid chromatography (HPLC). The effectiveness of this approach is demonstrated by chromatography of such glycopeptides obtained from the mucin populations, separated by isopycnic density-gradient centrifugation (figure 7B). The ion-exchange HPLC profiles indicate a clear separation between two major components and also identify a third species unresolved in the density gradient. In general, we have noted that there is a correlation between increasing density of the intact mucins in a CsCI density gradient and the charge density of the cognate glycopeptides as detected by elution position from the ionexchange column. Thus, the more highly sialylated and/or sulfated mucins have a higher buoyant density. Using this approach to investigate heterogeneity we have prepared high M, glycopeptides from the entire, unfractionated population of mucins isolated from "normal" airway secretions as well as from sputa from
0.6
Fig. 6. CsCIIO.2 M guanidiniumchloride density-gradient centrifugation of "normal"and bronchiectatic mucins.lsopycnic density-gradient centrifugation was performedon purified "normal"(opencirc/es) and bronchiectatic mucins (solid line)at a starting density of 1.52g/ml for approximately 90 h at 118,000 gay and 15° C.
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15 x 106) , population of molecules. Mucins from the fractionated population were pooled as "heavy" (hatched area) and "light" (clear area) fractions. (b) The glycopeptides prepared from these two fractions were chromatographed on a Mono a HR 5/5 column eluted with a linear gradient of zero to 0.25 M lithium perchlorate (dashed line). Aliquots from each fraction were analyzed with the periodic acid-Schiff procedure (see legend to figure 3).
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gests evidence for the presenceof three or four distinct species (63). These different populations are all large mucus glycoproteins with M, between 5 and 30 x 106 that form subunits after reduction and high M, glycopeptides after subsequent trypsin digestion. The subunits prepared from each of these mucin populations have similar reactivity with a polyclonal antiserum directed towards protein epitopes. Conclusion Our general observation is that respiratory mucus contains a heterogeneous mixture of mucus glycoproteins. Ion-exchange HPLC of the glycopeptides derived from the various species of mucins suggests that the molecules differ in the extent of sialylation and sulfation of their constituent oligosaccharides. We are at present studying respiratory mucin glycopeptides from a large number of subjects with different hypersecretoryconditions, including asthma, chronic bronchitis, and CF, in order to ascertain whether distinct mucins (as defined by conserved patterns of oligosaccharides) are shared by a range of subjects. If so, questions about their cellular origin and biologic function arise. From a more clinical point of view, the pattern of glycoproteins present may be informative of the state of lung damage, bacterial infection, or the effectiveness of medication. However, to exploit these possibilities, it will be necessary to devise probes specific to these components. Acknowledgment The writers thank Joy Greenwood, Marj Howard, and Helen Lindgren for technical assistance.
References Fig. 8. Example of analyses of mucin glycopeptidesafter ion-exchange HPLC. CF mucin glycopep,tides were chroHR 5/5 matographed on a Mono column and eluted with a linear gradient of zeroto 0.25M lithium perchlorate (gradient as in figure 7b). Fractions were analyzed for A28o , sialic acid, and hexose (carbazole) and, after immobilization onto nitrocellulose, were stained with the periodic acid Schiff (PAS),high iron diamine (HID), and alcian blue (pH, 2.5) procedures (63).
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2. Toremalm NG. The daily amount of tracheobronchial secretions in man. Acta Otolaryngol Suppl (Stockh) 1960;158:43-53. 3. Bhaskar KR, O'Sullivan DD, Seltzer A, Rossing TH, Drazen TM, Reid LM. Density gradient study of bronchial mucus aspirates from healthy volunteers (smokers and non-smokers) and from patients with tracheostomy. Exp Lung Res 1985; 9:289-308.
4. BhaskarKR, O'SullivanDD, Opaskar-Hincman H, Reid LM, Coles SJ. Density gradient analysis of secretions produced in vitro by human and canine airway mucosa: identification of lipids and proteoglycans in such secretions. Exp Lung Res 1985; 10:401-22.
PAS
o
1. Sleigh MA, Blake JR, Liron N. The propulsion of mucus by cilia. Am Rev Respir Dis 1988; 137:726-41.
30
40
50
60
70
Fraction number
subjects with a range of respiratory disorders. Many different procedures have been used in the analysis of the ion-exchange column fractions, including solution assays for hexoseand sialic acid as well as histochemical staining (e.g., alcian blue, periodic acid-Schiff, highiron diamine) after immobilization on nitrocellulose membranes (61-63). An example of
the type of data obtained is shown in figure 8 and indicates the presence of a number of components. This type of investigation has led us to conclude that there are a number of different mucus glycoproteins present in respiratory secretions. Indeed, a detailed study of the mucus glycoproteins from five patients with CF sug-
5. Thornton DJ, Davies JR, Kraayenbrink M, Richardson PS, Sheehan JK, Carlstedt I. Mucus glycoproteins from 'normal' human tracheobronchial secretion. Biochem J 1990; 265:179-86. 6. Richardson PS, Somerville M. Mucus and mucus-secreting cells. In: Barnes PB, Rodgers IW, Thomson NC, eds. Asthma: basic mechanisms and clinical management. London: Academic Press, 1988;163-85.
7. Davies JR, Gallagher JT, Richardson PS, Sheehan JK, Carlstedt I. Macromolecules in cat airway secretions. Biochem J 1991; 275:663-9. 8. Murlas C, Nadel JA, Basbaum CB. A morphometric analysis of the autonomic innervation of cat tracheal glands. J Auton Nerv Syst 1980; 2:23-37.
59
STRUCTURE AND HETEROGENEITY OF MUCINS
9. Partanen M, Laitinen A, Hervonen A, Toivanen M, Laitenen LA. Catecholamine and acetylcholinesterase-containing nerves in human lower respiratory tract. Histochemistry 1982; 76:175-88. 10. Gallagher JT, Kent PW, Passatore M, Phipps RJ, Richardson PS. The composition of tracheal mucus and the nervous control of its secretion in the cat. Proc R Soc Lond [BioI] 1975; 192:49-76. 11. Richardson PS, Webber SE... The control of mucous secretion in the airways by peptidergic mechanisms. Am Rev Respir Dis 1987; 136(Suppl: 72-6). 12. Kyle H, Widdicombe JG. The effects of peptides and mediators on mucus secretion rate and smooth muscle tone in the ferret trachea. Agents Actions 1987; 22:86-90. 13. Richardson PS, Phipps RJ, Balfre K, Hall RL. The role of mediators, irritants and allergensin causing mucin secretion from the trachea. In: Respiratory tract mucus. Ciba Foundation Symposium 54. Amsterdam: Elsevier, 1978; 111-31. 14. Adler KB, Hendley DD, Davis GS. Bacteria associated with obstructive pulmonary disease elaborate extracellular products that stimulate mucin secretion by explants of guinea pig airways. Am J Pathol 1986; 125:501-14. 15. Klinger JD, Tandler B, Liedtke CM, Boat TF. Proteinases of Pseudomonasaeruginosa evoke mucin releaseby tracheal epithelium. J Clin Invest 1984; 74:1669-78. 16. Phipps RJ, Richardson PS. The effects of irritation at various levels of the airway upon tracheal mucus secretion in the cat. J Physiol (Lond) 1976; 261:563-81. 17. Peatfield AC, Richardson PS. The action of dust in the airways on secretion into the trachea of the cat. J Physiol (Lond) 1983; 342:327-34. 18. Jeffrey PK, Reid LM. The respiratory mucus membrane. In: Brain JD, Proctor DF, Reid LM, eds. Respiratory defense mechanisms. New York: Marcel Dekker, 1977; 193-245. 19. Spicer SS, Schulte BA, Charkin LW. Ultrastructural and histochemical observations of respiratory epithelium and gland. Exp Lung Res 1983; 4:137-56. 20. Mazzuca M, Lhermitte M, Lafitte J-J, Roussel P. Use of lectins for detection of glycoconjugates in the glandular cells of human bronchial mucosa. 1 Histochem Cytochem 1982; 30:956-66. 21. Gupta R, Jentoft N, Jamieson AM, Blackwell 1. Structural analysis of purified tracheobronchial mucins. Biopolymers 1990; 29:347-55. 22. Chace KV, Flux M, Sachdev GP. Comparison of physiochemical properties of purified mucous glycoproteins isolated from respiratory secretions of cystic fibrosis and asthma patients. Biochemistry 1985; 24:7334-41. 23. FeldhoffPA, Bhavanandan VP, Davidson EA. Purification, properties and analysis of human asthmatic bronchial mucin. Biochemistry 1979; 18: 2430-6. 24. Roussel P, Lamblin G, Lhermitte M, et ale The complexity ofmucins. Biochimie 1988;70:1471-82. 25. Breg J, Van Halbeek H, Vliegenthart lFG, Lamblin G, Houvenaghel M-C, Roussel P. Structure of sialyl-oligosaccharides isolated from bronchial mucus glycoproteins of patients (blood group 0) suffering from cystic fibrosis. Eur 1 Biochem 1987; 168:57-68. 26. Breg J, Van Halbeek H, Vliegenthart JFG, Klein A, Lamblin G, Roussel P. Primary structure of neutral oligosaccharides. Part 2. Eur J Biochem 1988; 171:643-54. 27. Klein A, Lam blin G, Lhermitte M, Roussel P, Breg 1, Van HaU,eek H, Vliegenthart JFG. Primary structure of neutral oligosaccharides derived from respiratory mucus glycoproteins of a patient
suffering from bronchiectasis, determination by combination of 500mHz HNMR spectroscopy and quantitative sugar analysis. Part 1. Eur 1 Biochem 1988; 171:631-42. 28. Lamblin G, Boersma A, Klein A, Roussel P, Van Halbeek H, Vliegenthart JFG. Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. 1 Biol Chern 1984; 259:9051-8. 29. Lamblin G, Boersma A, Lhermitte M, et ale Further characterization by a combined high performance liquid chromatographwH-Nlvlk approach of the heterogeneity displayed by the neutral carbohydrate chains of human bronchial mucins. Eur J Biochem 1984; 143:227-36. 30. Carlstedt I, Lindgren H, Sheehan lK, Ulmsten U, Wingerup L. Isolation and characterization of human cervical-mucus glycoproteins. Biochern J 1983; 211:13-22. 31. Carlstedt I, Sheehan JK. Macromolecular proteins and polymeric structure of mucus glycoproteins. In: Nugent 1, O'Connor M, eds. Mucus and mucosa. Ciba Foundation Symposium 109. London: Pitman 1984; 157-72. 32. Thornton Dl, Sheehan JK, Lindgren H, Carlstedt I. Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural architecture. Biochem J 1991; 276:667-75. 33. SJomiany BL, Takagi A, Liau YH,lozwiak Z, Slomiany A. In vitro acylation of rat gastric mucus glycoprotein with 3Hpalmitic acid. 1 Biol Chern 1984; 259:11997-2000. 34. Slomiany A, Liau YH, Takagi A, Laszewicz W, Slomiany BL. Characterization of mucus glycoprotein fatty acyltransferase from gastric mucosa. 1 BioI Chern 1984; 259:13304-8. 35. Witas H, Sarosiek J, Aonu M, Murty VLN, Slomiany A, Slomiany BL. Lipids associated with rat small-intestinal mucus glycoprotein. Carbohydr Res 1983; 120:67-76. 36. Hansson GC, Sheehan JK, Carlstedt I. Only trace amounts of fatty acids are found in pure mucus glycoproteins. Arch Biochem Biophys 1988; 266:197-200. 37. Shankar V, Naziruddin B, ReyesDe La Rocha S, Sachdev GP. Evidence of hydrophobic domains in human respiratory mucins. Effect of sodium chloride on hydrophobic binding properties. Biochemistry 1990; 29:5856-64. 38. Woodward H, Horsey B, Bhavanandan VP, Davidson EA. Isolation, purification and properties of respiratory mucus glycoproteins. Biochemistry 1982; 21:694- 701. 39. Creeth JM, Bhaskar KR, Horton lR, Das I, Lopez-Vidriero M-T, Reid L. The separation and characterization of bronchial glycoproteins by density-gradient methods. Biochem J 1977; 167: 557-69. 40. Houdret N, LeTreut A, Lhermitte M, Lamblin G, Degand P, Roussel P. Comparative action of reducing agents on fibrillar human bronchial mucus under dissociating and nondissociating conditions. Biochim Biophys Acta 1981; 668:413-9. 41. Bhaskar KR, Reid L. Application of density gradient methods to study the composition of sol and gel phases of CF sputa and the isolation and characterisation of epithelial glycoprotein from the two phases. In: Sturgess J, ed. Perspectives in cystic fibrosis. Proceedings of the 8th Cystic Fibrosis Congress. Toronto: Imperial Press, 1980; 113-21. 42. Harding SE, Creeth JM. Self-association, polydispersity and thermodynamic non-ideality in a cystic fibrotic glycoprotein. IRCS Med Sci 1982; 10:474-5. 43. Sheehan JK, Carlstedt I. Size heterogeneity of human cervical-mucus glycoproteins. Studies
performed with rate-zonal centrifugation and laserlight scattering. Biochem 1 1987; 245:757-62. 44. Sheehan lK, Carlstedt I. Hydrodynamic properties of human cervical-mucus glycoproteins in 6M guanadinium hydrochloride. Biochem 1 1984; 217:93-101. 45. Carlstedt I, Sheehan JK. Structure and macromolecular properties of cervical mucus glycoproteins. In: Chantler E, Ratcliffe NA, eds. Mucus and related topics. SEB Symposium, 43. Cambridge: the Company of Biologists, 1989; 289-316. 46. Shogren RL, Jamieson AM, Blackwell J, 1entoft N. Conformation of mucous glycoproteins in aqueous solvents. Biopolymers 1986; 25:1505-17. 47. Jenssen AO, Harbitz 0, Smidsrod O. Electron microscopy of mucin from sputum in chronic obstructive bronchitis. Eur J Respir Dis 1980;61:71-6. 48. Harding SE, Rowe AJ, Creeth 1M. Further evidencefor a flexible and highly expanded spheroidal model for mucus glycoproteins in solution. Biochern J 1983; 209:893-6. 49. Rose MC, Voter WA, Brown CF, Kaufman B. Structural features of human tracheobronchial mucus glycoprotein. Biochem J 1984; 222:371-7. 50. Mikkelsen A, Stokke BT,Christensen BE, Elgasaeter A. Flexibility and length of human bronchial mucin studied using low-shear viscometry, birefringence relaxation analysis and electron microscopy. Biopolymers 1985; 24:1683-704. 51. Slayter HS, Lamblin G, LeTreutA, et ale Complex structure of human bronchial mucus glycoprotein. Eur 1 Biochem 1984; 142:209-18. 52. Sheehan lK, Oates K, Carlstedt I. Electron microscopy of cervical, gastric and bronchial mucus glycoproteins. Biochem J 1986; 239:147-53. 53. Marianne T, Perini J-M, Lafitte l-J, etal. Peptides of human bronchial mucus glycoproteins. Size determination by electron microscopy and by biosynthetic experiments. Biochem J 1987;248:189-95. 54. Mould AP, Holmes DF, Kadler KE, Chapman lA. Mica sandwich technique for preparing macromolecules for rotary shadowing. 1 Ultrastruct Res 1985; 91:66-76. 55. Lang D, Mitani M. Simplifiedquantitative electron microscopy of biopolymers. Biopolymers 1970; 9:373-9. 56. Koller T, Harford AG, Lee YK, Beer M. New methods for the preparation of nucleic acid molecules for electron microscopy.Micron 1969; 1:110-8. 57. Rose MC, Brown CF, Jacoby rz, Lynn WS, Kaufman B. Biochemical properties of tracheobronchial mucins from cystic fibrosis and non-cystic fibrosis individuals. Pediatr Res 1987; 22:545-51. 58. Carlstedt I, Lindgren H, Sheehan lK. The polymeric structure of human cervical-mucus glycoproteins. Studies of fragments obtained after reduction of disulphide bonds and after subsequent trypsin digestion. Biochem J 1983; 213: 427-35. 59. Sheehan lK, Boot-Handford RP, Chantler E, Carlstedt I, Thornton Dl. Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. Biochem 1 1991; 274:293-6. 60. Meselson M, Stahl FW, Vinograd J. Equilibrium sedimentation of macromolecules in density gradients. Proc Nat! Acad Sci USA 1957; 43:581. 61. Thornton nr, Holmes DF, Sheehan Kl, Carlstedt I. Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes. Anal Biochem 1989; 182:160-4. 62. Sheehan lK, Thornton nr, Carlstedt I. Histochemicalmethods used in biochemicalapproaches to mucus glycoproteins. Acta Histochem Suppl (Jena) 1990; 40:133-5. 63. Thornton nr, Sheehan lK, Carlstedt I. Heterogeneityof mucus glycoproteins from cysticfibrosis sputum. Biochem 1 1991; 276:677-82.
Introduction In the normal respiratory tract, inhaled matter such as dust, microorganisms, and chemical irritants is removed mainly by mucociliary transport. The coordinated beating of cilia propels an overlying layer of mucus toward the pharynx, where it is swallowed (1). In health, the amount of respiratory secretions reaching the trachea is very small (2); however, far greater amounts may be produced in conditions such as chronic bronchitis, asthma, and bronchiectasis. Airway mucus is a mixture of water, salts, protein, and high M, glycoconjugates. Mucus glycoproteins(mucins) are accepted as major constituents of mucus in hypersecretory conditions, but there is disagreement about the macromolecular composition of normal respiratory secretions. Bhaskar and coworkers (3, 4) have proposed that the main glycoconjugate from unstimulated healthy airways is proteoglycan, whereas we have observed mucus glycoprotein as the maj or secretory product and little if any proteoglycan (5). The mucus glycoproteinsconfer on mucus its characteristic properties of viscosity and elasticity, which enable it to be transported by cilia. Several cell types in the respiratory mucosa produce mucus glycoproteins, including the mucous cells in the submucosal glands and the goblet, and perhaps the ciliated, cells in the surface epithelium (6, 7). The control of respiratory mucus secretion is complex. The submucosal glands receive a dense innervation from the autonomic nervous system (8, 9), and both cholinergic and adrenergic agonists stimulate macromolecular output (10). In addition, neuropeptides (11), inflammatory mediators (12, 13),bacterial products (14, 15), and irritants (16, 17) have all been shown to modify mucus output. Histochemical studies (18, 19)have indicated the presence of different types of mucins, i.e., neutral and acidic (sulfated or sialylated), and these observations, as well as those made by using lectin histochemistry (20),suggest that different cells may produce structurally distinct mucins. Thus, respiratory mucus may contain a number of distinct populations of mucus glycoproteins and this, together with the complex control of secretion, presents scope for fine tuning of the response to a particular stimulus. However, there is as yet no biochemical evidence for different cellssynthesizing different mucins. The macromolecular structure of respiratory mucus glycoproteins has been the subject of some debate, and the recent literature indicates that opinions still differ as to the 54
SUMMARY Respiratory mucus glycoprotelns purified from both "normal" respiratory secretions and sputa of patients with a variety of hYPersecretoryconditions are high M, linear molecules adopting a random coil configuration in solution. Studies on their polydisperslty show them to have an M, In the range 3 to 32 X 106 and a distribution of length from 200 nm to beyond 10 J.1m. These macromolecules are fragmented by reduction of intermolecular disulfide bonds into subunits, with M, approximately 2 x 106 and length from 200 to 600 nm. Reduction not only cleaves the mucin molecule but opens, presumably by breaking Intramolecular disulfide bonds, cryptic "naked" protein regions. Trypsin digestion of subunits yields high M, glycopeptldes (M" 300 to 500,000), presumably by cleavage of the peptide core within the unfolded "naked" protein domains. Respiratory mucus glycoprotelns from infected sputum samples are usually heterogeneous In CsCI density gradients, In contrast to those from "normal" tracheobronchial secretions. The former are characterized by the presence of a number of different mucin species, and the basis for the separation of these mucins appears to be the variable presence of sialic acid and sulfate moieties In the oligosaccharide clusters. This heterogeneity may reflect a difference In cellular origin of the mucins and also may be clinically significant. AM REV RE8PIR DI8 1991; 144:84-89
size and heterogeneity of whole mucins as well as the existenceof a subunit structure for these molecules (21-24). However, it is agreed that proteolysis of these macromolecules yields a glycopeptide of M, 300 to 500,000, with a protein core rich in proline, serine, and threonine, which is densely substituted by a diverse range of O-linked oligosaccharides. These oligosaccharides have been the subject of detailed structural studies (25-29), but as to whether they are attached to distinct proteins or whether they have a particular organization along the protein core is not known.
Isolation/Purification In order to study the macromolecular properties of mucus glycoproteins, it is important to maintain, as far as possible, the integrity of the core protein throughout the isolation and purification process. Thus, high shear extraction methods and covalent bond-breaking reagents (i,e., dithiothreitol) should be avoided, and measures should be taken to suppress endogenous proteolytic activity in the samples. To this end we have adopted the procedure devised for the isolation and purification of cervical mucins (30, 31).In brief, the mucous gel is extracted with 6M-guanidinium chloride (GuHCI) in the presence of protease inhibitors. Insoluble material is removed by centrifugation, and the extractant is brought to 4M-GuHCI before the addition of CsCI to a final density of approximately 1.4g/ml. In our experience the gradient forming salt (typically CsCI) should not be added to the sample during extraction since the mucous gel may not be fully dispersed, and conventional high-speed centrifugation cannot then be employed to remove nonsolubilized material. Nonmucin proteins are removed by isopycnic centrifugation; two rounds are usu-
ally necessary for their complete removal, and a further purification step in CsCI/0.2MGuHCI removes contaminating DNA. This procedure works well even with heavily infected sputa in which large amounts of nonmucin protein and DNA are present (32). When working with sputum, enough mucin can be prepared from a single subject to permit a full characterization. So little mucus is produced by the healthy respiratory tract, however, that the main problem in studying these secretions lies in obtaining them in sufficient amounts from a single subject, and it is therefore necessary to pool secretions from a number of subjects (5).
Criteria for Purity We assay our samples by polyacrylamide gel electrophoresis with subsequent silver staining to reveal protein contamination and by analytical ultracentrifugation using A 2so monitoring for DNA. The specificabsorbance 1 From the Department of Biochemistry & Molecular Biology, University of Manchester, Manchester, United Kingdom, and the Department of Physiological Chemistry 2, University of Lund, Lund, Sweden. 2 Supported by the WellcomeTrust, UK; the Cystic Fibrosis Research Trust, UK; the Medical Research Council, UK; the British Lung Foundation; Grant No. 7902 from the SwedishMedical Research Council; the Swedish National Association against Heart and Chest Diseases; the National Swedish Environmental Protection Board; the Swedish Society for Cystic Fibrosis; Greta och Johan Kocks Stiftelser, Trelleborg, Sweden; the Medical Faculty, University of Lund, Sweden; and the National Swedish Board for Technical Development. 3 Correspondence and requests for reprints should be addressed to John K. Sheehan, Department of Biochemistry & Molecular Biology,University of Manchester, Manchester M13 9PT, UK.
55
STRUCTURE AND HETEROGENEITY OF MUCINS
for DNA is approximately 100 times that for mucins, making this a particularly sensitive assay. We estimate that contamination by nonmucin protein and DNA is in general below 1070 (wt/wt). It has been proposed that mucins contain a significant amount of lipid and also covalently bound fatty acids (33-35). However, when we prepared chronic bronchitic mucins by the procedure outlined above and extracted with organic solvents only small amounts of fatty acids (0.9 ug/rng of mucin) were detected, and no glycolipids, phospholipids, cholesterol, or triglycerides could be found (36). After lipid extraction, the mucins were subjected to alkaline methanolysis to release any covalently bound fatty acids, and the amount detected ranged from zero to 25 ng/mg of glycoprotein, Le., at the limits of detectability. We conclude that there are no detectable fatty acids covalently bound to respiratory or other mucins, though there are hydrophobic domains on these macromolecules (37), which may be responsible for the tight binding of fatty acids.
Size and Shape The size of respiratory mucus glycoproteins has been investigated by a number of workers, and a broad range of M, values have been reported (table 1). We have studied respiratory mucins from healthy human airways and from patients with asthma, cystic fibrosis (CF), or chronic bronchitis (5, 32). Values of M, were determined by using absolute-intensity, light-scattering methods (Zimm Plot analysis) and with sedimentation and diffusion measurements. A typical Zimm plot for a chronic bronchitic mucin is shown in figure 1 and yields values of M, and the radius of gyration (Rn) of 21 x 106 and 290 nm, respectively. Analytical ultracentrifugation indicates that the material is homogeneous although polydisperse in size with a marked concentrationdependent sedimentation rate (figure 2A). Quasi-elastic light-scattering measurements were performed to obtain the translational diffusion coefficient (Di), and extrapolation to both zero angle and concentration is necessary to obtain accurate values (figure 2B). We have obtained values of weight-average M, ranging from 14 to 25 x 106 for mucins isolated from both pooled "normal" respiratory secretions and chronic bronchitic sputum TABLE 1 SIZE OF RESPIRATORY MUCINS
Mr (x 10-6 )
Reference No.
3.5 1.5 2.3
22 23 38
CF sputum
9.3 17
21 32
Chronic bronchitic sputum
5-7 1.0
39 40
Source Asthmatic sputum
Normal secretions Definition of abbreviation: CF
14
= cystic
5 fibrosis.
10
Fig. 1. Zimm plot of light-scattering data for chronic bronchitic mucins. Readings were taken at 9 = 20,30,40,50, 60,75,90,110, and 130 degrees (closed circles) and extrapolation to 9 = 0 (open circles) was performed from 20 to 40 degrees. The concentrations used were 0.079,0.158,0.237, and 0.316mg/ml and extrapolation to zero concentration (open circles) yielded the radius of gyration. The optical constant was 9.32 x 10-8 cm 2g-2 •
.------------------~
8
6
4
2
O~--..!.---.....L..-----L.
0.0
0.5
1.0 . 2 Sin g/2
Fig. 2. The concentration-dependence of (a) the reciprocal sedimentation coefficient and (b) the translational diffusion coefficient for chronic bronchitic mucins. Sedimentation rate measurements (a) were performed on solutions with concentrations of 0.65, 0.864, 1.21,1.73,2.41, 3.46, and 4.81 mg/ml. The reciprocal sedimentation rate is plotted against concentration, and extrapolation to zero concentration was performed by linear regression with the use of least-squares analyses. The angular-dependence of the diffusion coefficient (Dt ) was performed on solutions (0.079,0.158,0.237, and 0.316 mg/ml), and extrapolation of Dt to zero angle was performed using values obtained at 9 = 20 to 40 degrees. The concentration-dependence of the diffusion coefficient at zero angle (b) is extrapolated to zero concentration by linear regression with the use of leastsquares analysis.
_ _---L_ _____l:....__-.J
1.5
2.0
2.5
3.0
+ 6000c
0.2
•
•
n I
0 r-
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~
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0.0
· 3
2
Concentration (mg/ml)
1.25
f
(/')
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E o
to
1.00 0
0 r-
x 0.75 0 .....
0.50 0.0
0.1
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0.3
Concentration (mg/ml)
(eight different subjects). In contrast, mucins isolated from CF sputum are of lower M, (32, 41); however, after gel-chromatography on Sepharose CL-2B the molecules excluded from the gel are similar in size to those found in normal secretions and chronic bronchitic sputa (32). In general, the values of M, obtained by us are higher than those reported by others (see table 1). This discrepancy may be accounted for by different preparation methods and sample handling. The values of Mr discussed above are averages for the whole distribution of molecules and tell us little about their range of mass and size.Equilibrium centrifugation (42), ratezonal centrifugation in GuHCI gradients (43), and gel chromatography on Sephacryl S-loo0 (21) are methods that can be used to investigate the polydispersity of macromolecules. A typical rate-zonal profile for CF respiratory mucins is shown in figure 3A. It is unlikely that the observed polydispersity is due to weak aggregation effects because fractions from the gradient (10 to 20 times more dilute) re-run close to their original position (figure 3B). Absolute-intensity, light-scattering measurements on fractions taken across the gradient yield values of M, in the range of 3 to 32 X 106 and Rn in the range of 100 to 490 nm.
The relationship between Ro and M, can be expressed as a Mark-Houwinck-type equation: Ro = kMa, where a is a shape-dependent parameter (44). A plot of log Ro against log M, (figure 3C) yields a line of slope a = 0.66, which is consistent with the mucins being linear and flexible molecules behaving as expanded random coils in solution. Similar findings have been reported for chronic bronchitic, pig gastric (45), CF respiratory (21), human cervical (44), and pig submaxillary (46) mucins. The architecture of respiratory mucins has been examined by a number of investigators using electron microscopy (47-53). There is agreement between these studies in that the molecules appear as flexible random coils, polydisperse in size, with lengths from 0.2 to 5 urn, However, the length distributions of the mucins vary between the different groups, presumably reflecting differences in sample purification and preparation for electron microscopy. Harding and coworkers (48), studying CF mucins and using a critical drying procedure, visualized the molecules as "beaded" structures with distinct globular domains occurring regularly along a filamentous thread. Rotary shadowing methods also have been used (49-51, 53). In these studies the mole-
56
SHEEHAN, THORNTON, SOMERVILLE, AND CARLSTEDT
75 50
J:;-
'enc 2c
"TI
2 2 O'l 2c
(l)
"TI
25
'L:
o
a ~_----L.-_-----L---,-------,-_._....I....-_----'-------1
5
...r:::
7
.~
u
E
100 75
c
U 6
50
'c 0
::J C)
25
5
11
13
15
Fraction number
2.5
2.0
6.5
7.0
7.5
Fig. 3. Polydispersity of CF mucins. In panel a, mucins (closed circles) were fractionated by using rate-zonal centrifugation on a guanidinium chloride gradient (described in references 5 and 43). The fractions were analyzed with the periodic acid-Schiff reaction after slotblotting onto a nitrocellulose membrane, and the color yield (integrated intensity) was quantitated with an image analysis system used as a reflectance densitometer (62). The concentration of guanidinium chloride (dashed line) was determined by refractive index measurements. In panel b, fractions 3 (closed circles), 8 (open circles), and 11(closed squares) from (a) were resubjected to rate-zonal centrifugation. In panel c, values of M r and RG across the mucin distribution of (a) were determined from absolute-intensity light-scattering data and are plotted as log-RG against log-Mr.
8.0
Fig. 4. Electron microscopy of "normal" respiratory mucins. The. macromolecules were spread in benzyldimethylalkylammonium chloride monolayers on a hypophase of (a) 6M-GuHCI and (b and c) 0.2 M NaCI, picked up on carboncoated grids, and made visible by rotary shadowing with platinum/tungsten (52).
culesweresprayed onto mica, and subsequently a replica was formed by shadowing with platinum and carbon. This technique, though applicable to most proteins, is not in general suitable for very large flexible molecules (i.e., mucus glycoproteins and DNA) where the high shear forces involved in atomizing the droplets may fragment such structures. For example, even triple-helical collagen molecules are sheared by this procedure (54). We have studied respiratory mucins using spreading techniques as employed in visualizing DNA (55, 56). The advantages of this approach are 2-fold. First, it avoids high shear forces during deposition of the molecules on the grid, and second, it is possible to deposit molecules in the presence of the same denaturing solvent (6M-GuHCI) that was used for characterizing the molecules in solution. The appearance of "normal" respiratory mucins, spread in 6M-GuHCI, is shown in figure 4A. The moleculesare convoluted,flexiblethreads, polydisperse in length, ranging from 300 nm to several microns, although their precise lengths are difficult to measure because of their tortuous nature and entanglement. The molecules are similar after spreading in 0.2 M NaCI, but they are less convoluted (figure 4B). In all solvent systems employed, there is evidence of heterogeneity, with a proportion (approximately 10070) of the molecules having a distinctly lower contrast (Le., thinner) than the rest (figure 4B). Mucins with a randomly meandering configuration typically have an uneven and "beaded" appearance, whereas those distorted under flow appear as smooth and thin filaments, often more than 10 urn in length (figure 4C). Thus, it appears that mucins have regions that are highly folded and condensed and that can be "unfolded" under the influence of mechanical forces. If true, this could have important consequences for the rheologic properties of a mucous gel where elastic energy may be stored within individual molecules as wellas the network of the gel.
Fragmentation of the Protein Core We and others (5, 21, 32, 39) find distinct changes in molecular size of respiratory mucins after reduction. In studies on "normal" human (5) and CF respiratory mucins (32), employing gel chromatography and electron microscopy, we have shown that the major fragment obtained after reduction of disulfide bonds is significantly smaller than whole mucins and is visualized as a linear flexible filament 200 to 600 nm in length. The chromatographic behavior of this fragment is similar to the reduced CF respiratory mucins isolated by Rose and coworkers (57), and it is thus very similar to subunits (M, 2 x 106 ) prepared from cervical mucins (58). Therefore, we propose that respiratory mucins are composed of subunits similar to those from cervical mucins. This is in agreement with the data of Gupta and coworkers (21) for CF respiratory mucins, but the existence of this subunit structure is not accepted by all workers in the field. For example, Chace and co-
57
STRUCTURE AND HETEROGENEITY OF MUCINS
workers (22), using light-scattering techniques, observed no decrease in M, (3.5 to 3.8 x 106 ) of asthmatic and CF respiratory mucins after reduction, whereas Roussel and coworkers (24) have suggested that the effects of reducing agents may be explained without involving the rupture of intermolecular disulfide bonds. Subunits, prepared from chronic bronchitic, gastric, and salivary mucins (59) as well as CF mucins (unpublished observation), crossreact with polyclonal antibodies directed towards "cryptic" protein domains exposed after reduction of whole cervicalmucins (59). These data indicate that respiratory mucins share protein epitopes with human cervical, gastric, and salivary mucins. Proteolysis of mucus glycoproteins releases a major population of large glycopeptides and smaller peptide and glycopeptide fragments, the latter probably originating from the "naked" protein regions on the core. The value of M, of the major glycopeptide fragment is dependent upon the protease employed (e.g., papain generates smaller molecules than does trypsin) and the degree of substitution and size of the oligosaccharides. Trypsin digestion of subunits generates smaller fragments than those obtained by direct digestion of mucins, and we distinguish between these situations by calling the former "T-domains" and the latter "tryptic glycopeptides." The high M, glycopeptides correspond to the dense "oligosaccharide clusters" found periodically along the molecule, and our studies on these structures indicate that they have a M, of 300 to 500,000, with lengths between 80 and 180 nm. A schematic diagram of the proposed mucin structure is shown in figure 5.
Heterogeneity Isopycnic centrifugation employed for the purification of respiratory mucins suggested to us the presence of different macromolecular species in many samples. Whereas "normal" mucins appeared as a homogeneous, almost Gaussian, distribution in the CsCI/ 0.2M-GuHCI density gradient, those from patients with CF (32) or other hypersecretory conditions (Somervilleand coworkers, unpublished observations) wereoften heterogeneous (figure 6). The resolution in an isopycnic density gradient is dependent on the M, of the molecules, the band width being related to the M, according to the relationship (60): 0 2 = RT PolM r(jj 2 rs (dp/dr), where Po is the initial solution density, 0 is the Y2 band width at 0.607 x peak height, co is the angular velocity of the rotor, rs is the distance from the rotor center to the band peak, and dp/dr is the density gradient at the band peak. Polydispersity of M, and nonideality effects caused by high concentration of the mucins, often a feature of preparative work, broaden density distribution profiles and obscure heterogeneity. To overcome this we have fractionated, by using rate-zonal centrifugation on GuHCI gradients, purified bronchiectatic mucins in order to obtain molecules (about 20070 of the total) with molecular
(a)
/~
Fig. 5. Schematic diagram of the proposed "architecture" of respiratory mucins. In panel (a), the whole mucins are made up of subunits joined end to end by disulfide bonds (s-s). (b) Upon reduction, subunits are released that contain alternatingoligosaccharideclustersand "naked" protein domains. Immunologic data suggest that the latter become unfolded by reduction (59). Trypsindigestion of subunits yields high Mr glycopeptides (T-domains) corresponding to the oligosaccharide clusters. These clustersare represented as "rodlike" only for the convenience of drawing. SU = freethiol generatedafter reduction of s-s.
~s-s)
/
SI
LU
S
I
"
(b) (.~ SH
trypsin ----~
v~
oligosaccharide 'cluster'
?
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'naked' stretch of protein weights in excess of 15 x 106 • When resubjected to density-gradient centrifugation in CsCI/0.2 M-GuHCI the mucins band more sharply, and the heterogeneity is more pronounced (figure 7A). The data confirm that the molecules are of high M«, which, if because of self-association, implies that aggregates must be very stable and specific, with the molecules not disposed to heterologous interactions. Although mucins may be fractionated into different species using this approach, it is impractical because of the small proportion of the mucin population utilized and the dilution required to achieve the fractionation. The observations on heterogeneity at the whole mucin level predict that the macromolecules are constructed from different types of "oligosaccharide clusters." These structures, which are a feature of all respiratory mucins we have studied, are smaller, more homogeneous in size, and thus more amenable to fractionation by high-resolution techniques
such as ion-exchange high performance liquid chromatography (HPLC). The effectiveness of this approach is demonstrated by chromatography of such glycopeptides obtained from the mucin populations, separated by isopycnic density-gradient centrifugation (figure 7B). The ion-exchange HPLC profiles indicate a clear separation between two major components and also identify a third species unresolved in the density gradient. In general, we have noted that there is a correlation between increasing density of the intact mucins in a CsCI density gradient and the charge density of the cognate glycopeptides as detected by elution position from the ionexchange column. Thus, the more highly sialylated and/or sulfated mucins have a higher buoyant density. Using this approach to investigate heterogeneity we have prepared high M, glycopeptides from the entire, unfractionated population of mucins isolated from "normal" airway secretions as well as from sputa from
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Fig. 6. CsCIIO.2 M guanidiniumchloride density-gradient centrifugation of "normal"and bronchiectatic mucins.lsopycnic density-gradient centrifugation was performedon purified "normal"(opencirc/es) and bronchiectatic mucins (solid line)at a starting density of 1.52g/ml for approximately 90 h at 118,000 gay and 15° C.
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15 x 106) , population of molecules. Mucins from the fractionated population were pooled as "heavy" (hatched area) and "light" (clear area) fractions. (b) The glycopeptides prepared from these two fractions were chromatographed on a Mono a HR 5/5 column eluted with a linear gradient of zero to 0.25 M lithium perchlorate (dashed line). Aliquots from each fraction were analyzed with the periodic acid-Schiff procedure (see legend to figure 3).
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gests evidence for the presenceof three or four distinct species (63). These different populations are all large mucus glycoproteins with M, between 5 and 30 x 106 that form subunits after reduction and high M, glycopeptides after subsequent trypsin digestion. The subunits prepared from each of these mucin populations have similar reactivity with a polyclonal antiserum directed towards protein epitopes. Conclusion Our general observation is that respiratory mucus contains a heterogeneous mixture of mucus glycoproteins. Ion-exchange HPLC of the glycopeptides derived from the various species of mucins suggests that the molecules differ in the extent of sialylation and sulfation of their constituent oligosaccharides. We are at present studying respiratory mucin glycopeptides from a large number of subjects with different hypersecretoryconditions, including asthma, chronic bronchitis, and CF, in order to ascertain whether distinct mucins (as defined by conserved patterns of oligosaccharides) are shared by a range of subjects. If so, questions about their cellular origin and biologic function arise. From a more clinical point of view, the pattern of glycoproteins present may be informative of the state of lung damage, bacterial infection, or the effectiveness of medication. However, to exploit these possibilities, it will be necessary to devise probes specific to these components. Acknowledgment The writers thank Joy Greenwood, Marj Howard, and Helen Lindgren for technical assistance.
References Fig. 8. Example of analyses of mucin glycopeptidesafter ion-exchange HPLC. CF mucin glycopep,tides were chroHR 5/5 matographed on a Mono column and eluted with a linear gradient of zeroto 0.25M lithium perchlorate (gradient as in figure 7b). Fractions were analyzed for A28o , sialic acid, and hexose (carbazole) and, after immobilization onto nitrocellulose, were stained with the periodic acid Schiff (PAS),high iron diamine (HID), and alcian blue (pH, 2.5) procedures (63).
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2. Toremalm NG. The daily amount of tracheobronchial secretions in man. Acta Otolaryngol Suppl (Stockh) 1960;158:43-53. 3. Bhaskar KR, O'Sullivan DD, Seltzer A, Rossing TH, Drazen TM, Reid LM. Density gradient study of bronchial mucus aspirates from healthy volunteers (smokers and non-smokers) and from patients with tracheostomy. Exp Lung Res 1985; 9:289-308.
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PAS
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1. Sleigh MA, Blake JR, Liron N. The propulsion of mucus by cilia. Am Rev Respir Dis 1988; 137:726-41.
30
40
50
60
70
Fraction number
subjects with a range of respiratory disorders. Many different procedures have been used in the analysis of the ion-exchange column fractions, including solution assays for hexoseand sialic acid as well as histochemical staining (e.g., alcian blue, periodic acid-Schiff, highiron diamine) after immobilization on nitrocellulose membranes (61-63). An example of
the type of data obtained is shown in figure 8 and indicates the presence of a number of components. This type of investigation has led us to conclude that there are a number of different mucus glycoproteins present in respiratory secretions. Indeed, a detailed study of the mucus glycoproteins from five patients with CF sug-
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