Isolation and characterization of mucin from the serum of cystic fibrosis patients.
Using monoclonal antibody 19-9, elevated levels of the sialyl Lea antigen (NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNac beta 1-3R) are detected in serum from most cystic fibrosis patients. We now report further characterization of the serum antigen and evidence that it is on a mucin glycoprotein and not on glycolipids. The antigen has an apparent molecular weight greater than 2 X 10(6) by gel filtration on Sephacryl S-400. On density gradient centrifugation, the antigen has a density of 1.54 g/ml in cesium chloride and 1.42 g/ml in cesium chloride/4 M guanidine HCl. Immunostaining with monoclonal antibody 19-9 of lipid extracts from cystic fibrosis patient serum and erythrocytes does not detect any antigen on glycolipids. The antigen was purified by gel filtration and density gradient centrifugation. After tritium labeling of the sialic acid residues, sodium dodecyl sulfate gel electrophoresis separates two subunits with apparent molecular weights of 200,000 and 400,000. All of the labeled sialic acid is released as low molecular weight oligosaccharides after mild alkaline borohydride degradation. The purified antigen contains fucose, galactosamine, glucosamine, and galactose but no mannose and is enriched in the amino acids threonine, serine, glycine, proline, and alanine. The purified antigen binds several antibodies recognizing epitopes common to many mucins. Thus, the physical, biochemical, and immunochemical properties of the purified antigen indicate that the sialyl Lea antigen is present on mucins in the serum of cystic fibrosis patients.
Roberts
D D
DD
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Rose
M C
MC
Wang
W T
WT
Chernick
M S
MS
Frates
R C
RC
D000293:Adolescent; D000328:Adult; D000596:Amino Acids / Q000032:analysis; D000941:Antigens / Q000097:blood; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002675:Child, Preschool; D002850:Chromatography, Gel; D003550:Cystic Fibrosis / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007118:Immunoassay; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009077:Mucins / Q000097:blood*
D000596:Amino Acids; D000941:Antigens; D002241:Carbohydrates; D009077:Mucins