The role of disulphide bonds in human intestinal mucin.
Goblet-cell mucin (mucin 1) was isolated and purified from human small-intestinal scrapings. After application of mucin 1 to DEAE-Bio-Gel (A) columns, most of the glycoprotein (76-94% of hexoses) was eluted in the first peak (designated mucin 2). Minor amounts of acidic glycoproteins were eluted with 0.2m- and 0.4m-NaCl in later peaks. Analyses of mucin 1 and mucin 2 revealed mucin 2 to be a monodisperse highly glycosylated glycoprotein containing 6.3% by wt. of protein, N-acetylgalactosamine, N-acetylglucosamine, galactose and fucose. Mucin 1 was similar in composition, but was polydisperse and contained more protein (12.3% by wt.) as well as N-acetylneuraminic acid. Analytical CsCl-gradient ultracentrifugation showed both mucin 1 and mucin 2 to have a major component with an average buoyant density of 1.47000g/ml. Mucin 1 also contained a slightly less-dense minor glycoprotein component. After exhaustive reduction and alkylation mucin 1 retained its major component, but partly dissociated into two lighter glycoprotein components. Mucin 2, in contrast, did not change its density distribution after reduction. Band ultracentrifugation in (2)H(2)O-containing iso-osmotic buffers showed that mucin 1 contained a major fast-sedimenting component (s(o)=37+/-2S), and a minor amount of a slower-sedimenting component. After reduction there was an increased quantity of the latter component, for which an s(o) value of 14.5S was calculated. In contrast, mucin 2 was unaltered by reduction (s(o)=33+/-2S). These findings indicate that the major component of goblet-cell mucin (mucin 2) does not dissociate after S-S-bond reduction, and thus does not apparently rely for its polymeric structure on the association of subunits through covalent disulphide bonds. However, the effects of reduction on mucin 1 suggest that in the native mucin intramolecular disulphide bonds in the minor glycoproteins may stabilize their structure, permitting secondary non-covalent interactions to develop with the major dense mucin (mucin 2) protein.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides / Q000502:physiology*; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007413:Intestinal Mucosa / Q000032:analysis*; D009077:Mucins
D000596:Amino Acids; D002241:Carbohydrates; D004220:Disulfides; D009077:Mucins; D004229:Dithiothreitol