Abstract
Highly purified human mucins from postmortem intestinal tissue were fractionated on anion exchange columns to generate separate neutral and acidic species. The neutral mucin (less than 1.0 mol % sialic acid) was the major species (greater than 80% by weight) and contained a higher molar proportion of fucose, galactose, and N-acetylglucosamine, and a lower proportion of sialic acid and N-acetylgalactosamine than the acidic species (greater than 10 mol % sialic acid). Amino acid analyses revealed a highly significant enrichment in serine, aspartate, and alanine in the neutral species and proline, threonine, and glycine in the acidic species. Thiol reduction of each species to remove their integral 118,000-dalton component did not alter the essential interspecies differences. Differences in threonine, proline, and serine also remained after removal of all "naked" or pronase-susceptible peptide regions from each species. These results indicate that neutral and acidic mucins contain glycopeptide segments exclusive of their 118,000-dalton and naked peptide components, which differ in amino acid composition. The key amino acid markers are similar to those observed for fuco- and sialoglycopeptides obtained after proteolytic digestion of human colonic mucin by Gold et al. (Gold, D.V., Schochat, D., and Miller, F. (1981) J. Biol. Chem. 256, 6354-6358). The oligosaccharide composition of small intestinal and colonic mucin may therefore depend upon transcriptional control of the synthesis of specific mucin peptides as well as the post-translational activity of glycosyltransferases. These findings may have significance for the quality and functions of mucus produced in a variety of pathological states.
Highlights
Purified human mucins from postmortem in- another discrete peptide, which is enriched in cysteine and testinal tissue were fractionated on anion exchange performs the function of linking the large glycopeptides tocolumns to generate separate neutral and acidic spe- gether by disulfide bridges
Differences in threonine,proline, and serine native mucin, thereare at least two different regions of remained after removal of all “naked” or pronase- peptide: (a)a “naked” segment(s) containinngo carbohydrate, susceptible peptide regions from each species. These sensitive to attack by proteases and, in the case of human results indicatethat neutral andacidic mucins contain intestinal mucin, bearing the major antigenic determinants of glycopeptide segments exclusive of their 118,000-dalton and naked peptide components, which differ in amino acid composition
Proteoglycans, and care was taken during preparation and Many of the mucin samples were derived from patients handling to with cystic fibrosis, a disease in which mucus is notably avoid proteolytic degradation
Summary
118,000-daltonlink glycopeptideor the naked peptide regions in the mucin. The differences appear to be localized to thehighly glycosylated regionof peptide in thelarge mucin glycopeptides.We speculate that these findings mayhave. University of Calgary,Health Sciences Centre, Calgary,Alberta, Canada. ’The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, II Present address: Genetics Laboratory, St. Christopher’s Hospital, polyacrylamide gel electrophoresis; ECTEOLA, epichlorohydrin tri-. Christopher’s Hospital, polyacrylamide gel electrophoresis; ECTEOLA, epichlorohydrin tri- Neutral and Acidic Species of Mucin significance for the biosynthesis and physical properties of (w/v) NaN3). The enzyme was added at 0, 24, and 48 h to give a final intestinal mucins in health and disease. Digests were applied to Sepharose 4B (1.5 X 46 cm), and the column was eluted with 0.2 M
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