BACKGROUND/AIMS: Human colonic epithelium produces large amounts of mucin. The aim of this study was to examine mucin biosynthesis in the human colon. METHODS: Human colonic mucin was isolated using CsCl density gradients, and polyclonal antiserum was raised. Biosynthesis of colonic mucins was studied by labeling colonic explants with 35S-labeled amino acids or [35S]sulfate and subsequent immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The polyclonal antiserum specifically recognized colonic mucin, primarily reacting with peptide epitopes. Biosynthetic pulse/chase experiments showed a 35S-amino acid-labeled mucin precursor of about 600 kilodaltons, which was converted into a mature, glycosylated, and sulfated mucin and subsequently secreted into the medium. This mature mucin comigrated with isolated colonic mucin with an apparent molecular weight of 550 kilodaltons on SDS-PAGE, whereas gel filtration indicated that the molecular weight is actually much larger. Independent immunoprecipitation with an anti-Muc2 antiserum showed cross-reactivity with the 600-kilodalton precursor. CONCLUSIONS: These results show the biosynthesis of a secretory colonic mucin for the first time. This mucin is synthesized as a precursor protein of approximately 600 kilodaltons, which, after glycosylation, is secreted as a glycoprotein with an apparent molecular weight of 550 kilodaltons on SDS-PAGE. It is very likely that this mucin is Muc2.
BACKGROUND/AIMS: Human colonic epithelium produces large amounts of mucin. The aim of this study was to examine mucin biosynthesis in the human colon. METHODS:Human colonic mucin was isolated using CsCl density gradients, and polyclonal antiserum was raised. Biosynthesis of colonic mucins was studied by labeling colonic explants with 35S-labeled amino acids or [35S]sulfate and subsequent immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The polyclonal antiserum specifically recognized colonic mucin, primarily reacting with peptide epitopes. Biosynthetic pulse/chase experiments showed a 35S-amino acid-labeled mucin precursor of about 600 kilodaltons, which was converted into a mature, glycosylated, and sulfated mucin and subsequently secreted into the medium. This mature mucin comigrated with isolated colonic mucin with an apparent molecular weight of 550 kilodaltons on SDS-PAGE, whereas gel filtration indicated that the molecular weight is actually much larger. Independent immunoprecipitation with an anti-Muc2 antiserum showed cross-reactivity with the 600-kilodalton precursor. CONCLUSIONS: These results show the biosynthesis of a secretory colonic mucin for the first time. This mucin is synthesized as a precursor protein of approximately 600 kilodaltons, which, after glycosylation, is secreted as a glycoprotein with an apparent molecular weight of 550 kilodaltons on SDS-PAGE. It is very likely that this mucin is Muc2.
Authors: Mireille K Makkink; Nicole M J Schwerbrock; Michael Mähler; Jos A Boshuizen; Ingrid B Renes; Markus Cornberg; Hans J Hedrich; Alexandra W C Einerhand; Hans A Büller; Siegfried Wagner; Marie-Luise Enss; Jan Dekker Journal: Dig Dis Sci Date: 2002-10 Impact factor: 3.199
Authors: Thaddeus S Stappenbeck; John D Rioux; Atsushi Mizoguchi; Tatsuya Saitoh; Alan Huett; Arlette Darfeuille-Michaud; Tom Wileman; Noboru Mizushima; Simon Carding; Shizuo Akira; Miles Parkes; Ramnik J Xavier Journal: Autophagy Date: 2011-04-01 Impact factor: 16.016