Dataset Information

Effects of hydrogen peroxide, mild trypsin digestion and partial reduction on rat intestinal mucin and its disulphide-bound 118 kDa glycoprotein.

ABSTRACT: The role of the disulphide-bound 118 kDa glycoprotein of rat intestinal mucin is unknown, although it has been proposed to serve as a 'link' component for the mucin monomers. The present studies investigated release or destruction of the 118 kDa glycoprotein (monitored by gel electrophoresis and Western-blot analysis) during progressive breakdown of the mucin polymer (assessed by Sepharose 2B chromatography). H2O2 gradually destroyed the 118 kDa glycoprotein and dissociated the mucin polymer into components of similar size to the monomers. After 3 h, mucin samples contained almost no 118 kDa glycoprotein or its breakdown products, but 50% of the mucin was still eluted in the void volume of a Sepharose 2B column. Although mild trypsinolysis had little effect on the Sepharose 2B elution profile of the mucin, the 118 kDa glycoprotein was completely cleaved into 54-56 kDa and 60-66 kDa fragments which remained disulphide-bound to the high-molecular-mass mucin. Increasing levels of thiol reduction resulted in progressive loss of disulphide bonds, release of the 118 kDa glycoprotein and depolymerization of the mucin. Although approx. 40% of the mucin in partially reduced samples was recovered in the Sepharose 2B void volume, this material contained no 118 kDa glycoprotein and apparently consisted of disulphide-bound mucin monomers. Thus the 118 kDa glycoprotein may be destroyed by H2O2, extensively cleaved by trypsin or released by reduction without completely dissociating the mucin into monomers. Therefore the 118 kDa glycoprotein may not function as a 'link' component for all of the mucin monomers in the native polymer.

SUBMITTER: Mantle M

PROVIDER: S-EPMC1149965 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:1. A specific antibody was developed against the disulphide-bound 118 kDa glycoprotein of human intestinal mucin and used to establish an e.l.i.s.a. Fourteen purified mucins [eight normal (N) and six cystic fibrosis (CF)] had the same affinity for the antibody in the e.l.i.s.a., but their relative immunoreactivities varied widely (approx. 100,000-fold). In general, CF mucins were more antigenic than N mucins. 2. Variations (approx. 10-fold) were detected in the 118 kDa glycoprotein content of both N and CF mucins (assessed from Coomassie Blue-stained polyacrylamide gels), but these did not appear to be responsible for the differences in mucin immunoreactivity. 3. Variations (approx. 6-fold) were also observed in the size of the 118 kDa peak produced by N and CF mucins on Western blots. These were mostly due to differences in the 118 kDa glycoprotein content of mucins, although a small proportion resulted from changes in the number of antigenic determinants within individual 118 kDa glycoproteins. 4. After concanavalin A affinity chromatography of four reduced mucins (two N and two CF), purified 118 kDa glycoprotein was recovered in the bound fractions from the column, specifically eluted by methyl alpha-mannoside. 5. The amounts of 118 kDa glycoprotein isolated from the four mucins varied as predicted from the size of their 118 kDa bands on Coomassie Blue-stained gels. 6. Three 118 kDa glycoproteins (one N and two CF) showed almost identical reactivity in the e.l.i.s.a.; the fourth had fewer antigenic determinants. 7. Since differences in 118 kDa glycoprotein content and in the number of antigenic determinants within the 118 kDa glycoprotein did not account for variations in the reactivity of native mucins in the e.l.i.s.a., it appeared that accessibility of the 118 kDa glycoprotein to antibody binding may be critical in determining mucin immunoreactivity. This suggests that the three-dimensional conformation of CF mucins may differ from that of N mucins, leading to increased antigenicity.

Dataset Information

Effects of hydrogen peroxide, mild trypsin digestion and partial reduction on rat intestinal mucin and its disulphide-bound 118 kDa glycoprotein.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets