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Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis airway secretions.

ABSTRACT: To investigate the genetic identities of the mucins secreted in cystic fibrosis (CF) airways, sputum was collected from five individuals. Samples were separated into gel and sol phases by high-speed centrifugation and the gel phase was extracted in 6 M guanidinium chloride. The 'insoluble' residue remaining after extraction of the gel phase was brought into solution by reduction/alkylation. Density-gradient centrifugation in CsCl revealed polydisperse distributions of sialic acid-containing mucins in the gel phase, insoluble residue and sol phase fractions and the degree of variation between the different individuals was low. Antibodies recognizing MUC5AC and MUC5B identified these mucins in each of the fractions. MUC2, however, was present only as a component of the insoluble residue from the gel which accounted for less than 4% by mass of the total mucins. MUC5B and MUC5AC from the gel phase were large oligomeric species composed of disulphide-bond linked subunits and MUC5B was present as two populations with different charge densities which are likely to correspond to MUC5B 'glycoforms'. The sol phase contained, in addition to MUC5AC and MUC5B, mainly smaller mucins which did not react with the antisera and which were probably degraded. MUC5AC appeared to be enriched in the sol, suggesting that this mucin may be more susceptible to proteolytic degradation than MUC5B. The mucins present in sputum remained broadly similar during acute exacerbation and following antibiotic treatment, although the relative amount of an acidic MUC5B glycoform was decreased during infection.

SUBMITTER: Davies JR

PROVIDER: S-EPMC1220647 | biostudies-other | 1999 Dec

REPOSITORIES: biostudies-other

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Project description:Mucous secretions were obtained from cat tracheas that had received [3H]glucose and [35S]sulphate to radiolabel mucus glycoproteins biosynthetically. Samples were collected under resting ('basal') conditions as well as after pilocarpine stimulation and were separated into gel and sol phases by centrifugation. Macromolecules were partially purified by using gel chromatography on Sepharose CL-4B, and the species that were eluted with the void volume were then separated into two major populations with isopycnic density-gradient centrifugation in CsCl. The major component from the gel phase of pilocarpine-induced secretions had a buoyant density typical of mucins and was observed as linear and apparently flexible chains by electron microscopy. Reduction of disulphide bonds gave subunits that could be further cleaved by trypsin digestion into components of approximately the same size as the high-Mr glycopeptides obtained from other mucins after this treatment. In contrast, the dominant species in the gel phase of the 'basal' secretion had a significantly higher buoyant density than expected for mucins and was largely unaffected by reduction, as studied by gel chromatography. The macromolecules were fragmented by trypsin, suggesting that they contain a polypeptide backbone. This more dense component also predominated in the sol phase both from the 'basal' secretions and from the pilocarpine-released secretions. Digestion with DNAase, chondroitin ABC lyase or heparan sulphate lyase had no effect, which shows that this component is not DNA, a dermatan sulphate/chondroitin sulphate or a heparan sulphate proteoglycan. In contrast, endo-beta-galactosidase and keratanase caused some fragmentation, suggesting that the molecules contain some linkages of the poly-(N-acetyl-lactosamine) type, although the degradation was not as extensive as expected for keratan sulphate. Treatment with alkaline borohydride resulted in extensive fragmentation of the high-Mr glycopeptides from both components, indicating that the glycans were oligosaccharides that were probably O-linked. The monosaccharide compositions of both components were consistent with that expected for mucins. The data are in keeping with the major component from the pilocarpine-stimulated gel secretions being a mucus glycoprotein and the more dense component being a mucin-like molecule, possibly related to the keratanase-sensitive material isolated from canine trachea by Varsano, Basbaum, Forsberg, Borson, Caughey & Nadel [(1987) Exp. Lung Res. 13, 157-184].

Dataset Information

Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis airway secretions.

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