Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions

Abstract

SMG (submucosal gland) secretions are a major component of the airway surface liquid, are associated with innate immunity in the lung, and have been reported to be altered in lung disease. Changes in lung mucosal glycosylation have been reported in CF (cystic fibrosis), which may be responsible for differential bacterial binding to glycosylated components in the lung mucosa and hence increased pre-disposition to pulmonary infection. Glycoproteomic analysis was performed on SMG secretions collected from explanted bronchial tissue of subjects with severe lung disease, with and without CF, and controls without lung disease. Mucins MUC5B and MUC5AC were shown to be the dominant high-molecular-mass glycoprotein components, with a minor non-mucin glycoprotein component, gp-340, also present. Oligosaccharides containing blood-group determinants corresponding to subjects' blood type were abundant on MUC5B/MUC5AC, as were Lewis-type epitopes and their sialylated analogues, which are ligands for pathogens and leucocytes. No significant differences were found in the glycosylation of MUC5B/MUC5AC or gp-340 between CF and non-CF subjects with severe lung disease, implying that CF does not influence SMG secretion mucin glycosylation in end-stage lung disease. There were also no significant differences found in the glycosylation of these components in severe lung disease compared with non-diseased lungs. This suggests that previously reported changes in the glycosylation of respiratory glycoconjugates in CF, and other pulmonary conditions, are not due to the glycosylation of components in SMG secretions, but may involve other secretions, responses or extracellular factors.

Figure 1. High-molecular-mass glycoproteins in SMG secretions collected from explanted bronchial tissue from non-CF and CF subjects with lung disease and controls

SMG secretions were collected from human explanted bronchial tissue, reduced and alkylated, separated by 1D SDS/Ag/PAGE, electroblotted to PVDF membrane and (A) stained for acidic oligosaccharides with Alcian Blue. All samples contained major components at ≈4 MDa, with minor components present down to ≈1 MDa. (B) Normalized intensity of Alcian Blue staining down the molecular-mass range containing glycoproteins. Error bars show S.E.M. values.

Figure 2. MALDI-TOF-MS spectra of tryptic digests from SMG secretions from explanted bronchial tissue

Major protein components were excised from the PVDF membrane, digested with trypsin, and the resulting peptide mixture was analysed by MALDI-TOF-MS. (A) Spectrum from the ≈4 MDa protein band from CF subject 1. Peptide ions shown in boldface, MUC5B peptides; peptide ions shown in italics, MUC5AC peptides; Trypsin, autodigest peak; ACTH (adrenocorticotropic hormone), internal calibration peptide. (B) Spectrum from the ≈1 MDa protein band from non-CF subject 3. Bold peptide ions, gp-340 peptides. Spectra are typical of all subjects.

Figure 3. Monosaccharide compositions of O-linked oligosaccharides from MUC5B/MUC5AC and gp-340 in SMG secretions from non-CF and CF subjects with lung disease and controls

The gp-340 containing band at ≈1 MDa and the MUC5B/MUC5AC containing band at ≈4 MDa were excised and O-linked oligosaccharides were released by reductive β-elimination. The resulting oligosaccharide alditols were analysed by LC-ESI-MS, and their relative intensities were determined based on single-ion chromatograms. GlycoComp™ was used to determine the monosaccharide composition of the detected oligosaccharides. Weighted average monosaccharide compositions, with error bars representing S.E.M. values, and typical MS spectra, are shown for (A and B) MUC5B/MUC5AC and (C and D) gp-340. MUC5B/MUC5AC was found to have a glycosylation pattern different from that of gp-340 variant protein, specifically with regard to fucosylation and sialylation, but there were no significant differences between CF and non-CF subjects with lung disease or controls. Abbreviations: HexNAc, N-acetylhexosamine; Hex, hexose; Fuc, fucose; NeuAc, N-acetylneuraminic acid; Sulf, sulphate.

Figure 4. Example of structural characterization of typical O-linked oligosaccharides from MUC5B/MUC5AC in SMG secretions by MS/MS

Oligosaccharides were released by reductive β-elimination from high-molecular-mass glycoproteins in SMG secretions from non-CF subject 4 and analysed by LC-ESI-MS/MS. Structures were assigned from fragment ions using the GlycosidIQ™ glycan mass-fingerprinting tool. (A) Example of a sialyl-Le-type structure. (B) Example of blood-group A/Le type structure. Abbreviations: Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; GalNAcol, N-acetylgalactosaminitol; Fuc, fucose; NeuAc, N-acetylneuraminic acid; a, α linkage; b, β linkage; 1, 2, 3, 4, 6, linked carbon position.

Figure 5. Epitopes and substructures of O-linked oligosaccharides from MUC5B/MUC5AC in SMG secretions from explanted bronchial tissue from CF and non-CF subjects with lung disease and controls

The weighted average abundances of each structural epitope in oligosaccharides from MUC5B/MUC5AC (Supplementary Table S5; http://www.BiochemJ.org/bj/387/bj3870911add.htm) are shown as bar charts, with error bars representing S.E.M. values. There were no significant differences between CF lung disease, non-CF lung disease or control subjects in any of the structural epitopes detected. The occurrences of previously characterized oligosaccharides from human respiratory glycoproteins containing these epitopes were determined from GlycoSuiteDB® and are shown as pie charts. (A) Oligosaccharide charge: neutral, non-sialylated and non-sulphated; acidic, sialylated or sulphated. (B) O-linked core type (1, 2, 3 or 4). (C) N-Acetyl-lactosamine chain elongation type (1 or 2). (D) Terminal epitopes: Le a/x, Le^a or Le^x; Le b/y, Le^b or Le^y; Si Le a/x, sialyl-Le^a or sialyl-Le^x; Su Le a/x, sulpho-Le^a or sulpho-Le^x; A,B,H, blood-group A, B or H (O) antigen; Si Gal, sialylated galactose; Si GalNAc, sialylated core N-acetylgalactosamine; Su Gal, sulphated galactose. Abbreviations: Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAcol, N-acetylgalactosaminitol; Fuc, fucose; NeuAc, N-acetylneuraminic acid; a, α linkage; b, β linkage; 1, 2, 3, 4, 6, linked carbon position.