alcian-blue and Cystic-Fibrosis

The major phenotype of CF is the accumulation of mucus, a phenomenon whose relation to the dysfunctional CFTR is still not fully understood. This means that studies of mucus and its main component, the mucins, are important. Due to the large size and high glycosylation level, such questions need special considerations and methodology. We describe methods for the general quantification of heavily glycosylated proteins as the mucins using dot/slot blot. We also describe the separation of the mucins by gel electrophoresis and the identification with specific antibodies on Western blot and by proteomics.

Topics: Alcian Blue; Animals; Antibodies; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophoresis, Polyacrylamide Gel; Gene Expression; Glycosylation; Humans; Immunoblotting; Mice; Molecular Biology; Mucins; Mucociliary Clearance; Mucus; Proteomics; Solubility; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin

Defective acidification of intracellular organelles, particularly the trans-Golgi network, has been proposed to explain the decreased sialylation and increased sulphation of secreted proteins in cystic fibrosis (CF). To test this hypothesis we compared expression of sulphate and sialic acid on three salivary mucins namely MG1 (MUC-5B), MG2 (MUC-7) and GL. Proteins in whole mouth saliva (WMS) from four individuals were separated by fast protein liquid chromatography (FPLC) on a Superdex 200 column and the partially purified mucins slot-blotted and assayed for sulphate content by staining with Alcian Blue. Sulphation varied with the individual and with the mucin: MG1 was the most sulphated and contributed almost the entire sulphate content of WMS. These results allowed us to test small volumes of WMS from 20 CF patients and age- and sex-matched controls for estimates of sulphate content on MG1. Wherever possible sulphate on MG1 was also visualised by staining washed SDS-PAGE gels with Alcian Blue at pH 1.0. To assess the sialic acid content of salivary mucins, electroblots of SDS-PAGE gels were probed with labelled Triticum vulgaris agglutinin. In summary, our results show MG1 to be the main sulphated protein in whole mouth saliva and there are large differences in the expression of sulphate and of sialic acid on this mucin, both in control and CF groups. CF led neither a decrease in sialylation nor an increase in sulphation and direct comparisons of sialic acid content with sulphate in MG1 failed to reveal any obvious link between the two in health or in disease. Our data thus do not support the hypothesis of defective acidification as the underlying cause of altered glycosylation in CF, but point instead to inter-individual differences in expression/functioning of terminal glycosyltransferases for published observations. We thank the European Union Biomed II programme for support.

Topics: Acids; Alcian Blue; Coloring Agents; Cystic Fibrosis; Humans; Mucin-5B; Mucins; N-Acetylneuraminic Acid; Saliva; Salivary Proteins and Peptides; Sulfates; Wheat Germ Agglutinins