At least eight mucin apoproteins are expressed by the tracheobronchial epithelium, but it is not known which, if any, of these are major constituents of the respiratory secretions responsible for the formation of the mucus gel. To address this we have isolated mucins from normal, asthmatic and chronic bronchitic secretions. The asthmatic mucin reduced subunits were fractionated into four populations (I-IV) by anion-exchange HPLC. Amino acid and monosaccharide compositional analysis, as well as M(r) and size measurements, indicate that two of these populations (I and II) are glycoforms of the same or related apoprotein(s) and that the other populations contain two different apoproteins. A panel of antibodies and antisera recognizing the variable number tandem repeat (VNTR) of specific mucin apoproteins did not, as predicted, react with the glycosylated molecules, but after deglycosylation the majority of these probes (with the exception of those to MUC2, which were negative) reacted at a low level with each of the subunit populations. In contrast, an antiserum against a non-VNTR sequence of MUC5AC identified one of the populations (III) as the MUC5AC mucin. The MUC5AC reduced subunit had an M(r) of 2.2 x 10(6) and an RG (radius of gyration) of 57 nm. The genetic identities of the major mucin (populations I and II) and a minor component (population IV) were not established. The MUC5AC mucin was also identified as a major component in the pooled normal secretions from 20 individuals, whereas in a chronic bronchitic sample it was only a minor constituent. Furthermore, in all these different respiratory secretions the MUC5AC mucin appears as a similar biochemical entity, as assessed by Mono Q chromatography and agarose electrophoresis, suggesting that it may have a well-defined pattern of glycosylation in the respiratory tract.

The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.

Airway mucus from asthmatics is often unusually solid. The death of a patient in status asthmaticus allowed the collection of 28 g of abnormal airway mucus at autopsy. Its chemical and physical properties were studied to reveal differences from more normal airway mucus. The gel plug taken from the airways could be dispersed in 6 M guanidinium chloride, but it took > 1 wk and 700 ml of extractant to disperse 3 g of exudate completely. In contrast, treatment with 10 mM dithiothreitol, which reduces disulfide bonds, dispersed the gel within seconds. Mucins accounted for 25% of the non-dialyzable material in the gel, while DNA constituted < 1% and proteoglycans could not be detected. The mucins were similar in architecture and general composition to other respiratory mucins and were present at a high concentration (approximately 40 mg/ml). The majority of mucins were of extreme size (mean M(r) 30-40 x 10(6)) and slow to dissolve, but sequential extraction experiments on the gel exudate demonstrated a proportion of mucins (15%), the most readily extracted, which had a higher density, 1.45-1.55 g/ml, a lower M(r) (11.5 x 10(6)) and were markedly more acidic than the bulk of the mucins. Both major and minor mucin populations were extremely heterogeneous in mass distribution. Electron microscopy of the major mucin species demonstrated extensive networks of molecules many microns in length. The major mucin species was distinctly less acidic than mucins previously described from either normal or diseased airways. Amino acid analysis of fractions across the charge distribution suggested the presence of at least two different mucin proteins occurring as distinct glycoforms.

Background: Mucin polymer formation is a complex intracellular process.Results: MUC5B N-terminal D3-domains form reversible pH-sensitive calcium mediated cross-links between linear MUC5B polymer chains.Conclusion: Intracellular assembly of MUC5B generates disulfide-bonded polymers which form calcium mediated condensed networks in secretory granules.Significance: This identifies a new model for mucin assembly that may be common to other polymeric mucins.