Purification and characterization of sialyl-Le(a)-carrying mucins of human bile; evidence for the presence of MUC1 and MUC3 apoproteins.
Purification of sialyl-Le(a)-carrying mucins from primary human bile by trichloroacetic acid precipitation, delipidation, and gel filtration in guanidinium chloride gave three separable fractions, one of which was further purified by affinity chromatography. These fractions, named SBG1 (for soluble bile glycoprotein), SBG2, and SBG3 had molecular masses of > 1100, 800-950, and 100-250 kDa, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their mucin characteristics were indicated by a high carbohydrate content, ranging from 74 to 95%. The carbohydrate compositions indicated the presence of very long fucosylated polylactosamine chains. Amino acid analyses showed high abundance of serine and threonine in all three fractions (19-36%), confirming their mucin-like nature. Immunochemical analyses of deglycosylated samples detected the MUC1 mucin apoprotein in SBG2 and the MUC3 protein in SBG1. To our knowledge, this is the first report of a MUC3 mucin being purified. This mucin showed no significant reduction in size upon trypsin treatment or disulfide bond reduction and alkylation. Gel filtration of three samples of secondary bile showed that the size distribution of sialyl-Le(a)-carrying glycoproteins was similar to that found in primary bile, and immunochemical analysis showed that the MUC1 protein was present in all three samples. In one sample an additional fraction was isolated, which was insoluble in 6 M guanidinium chloride, but was solubilized upon reduction and alkylation. mRNAs from gallbladder epithelia were analyzed in Northern blot hybridizations showing that the MUC1 and MUC3 but not the MUC2 mucin apoprotein genes were expressed.
Department of Medical Biochemistry, University of Göteborg, Gothenburg, Sweden.
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D
Baeckström
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N
Karlsson
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GC
Hansson
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D001059:Apoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D001646:Bile / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000032:analysis; D015152:Blotting, Northern; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D005704:Gallbladder / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D018396:Mucin-1; D055262:Mucin-2; D009077:Mucins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012794:Sialic Acids / Q000032:analysis
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001059:Apoproteins; D014408:Biomarkers, Tumor; D006023:Glycoproteins; C524625:MUC2 protein, human; D008562:Membrane Glycoproteins; D018396:Mucin-1; D055262:Mucin-2; D009077:Mucins; D009844:Oligosaccharides; D012333:RNA, Messenger; D012794:Sialic Acids; C058981:sialyl-Le(a) oligosaccharide