The existence of a discrete ‘link’ peptide in epithelial mucins has been debated for many years. There is evidence that at least some mucins contain a specific ‘link’ peptide (or glycopeptide) that enhances mucin polymerization by forming disulphide bridges to large mucin glycoprotein subunits. A major difficulty has been to know whether the reported differences in putative ‘link’ components represent artifacts generated by inter-laboratory differences in technical procedures used in mucin purification. The present paper outlines the results of a collaborative study involving five laboratories and 53 samples of purified gastrointestinal mucins (including salivary, gastric, small-intestinal and colonic mucins) prepared by five techniques from four different animal species. An early step in mucin purification in all cases was the addition of proteinase inhibitors. Representative mucins were analysed for their composition, electrophoretic mobility in SDS/polyacrylamide-gel electrophoresis before and after disulphide-bond reduction, and for their reactivity with monospecific antibodies developed against the 118 kDa putative ‘link’ glycopeptide isolated from either rat or human small-intestinal mucins. Our results indicate that, despite differences in laboratory techniques, preparative procedures, organs and species, each of the purified mucins contained a ‘link’ component that was released by disulphide-bond reduction and produced a band on SDS/polyacrylamide-gel electrophoresis at a position of approx. 118 kDa. After electroelution and analyses, the 118 kDa bands from the different mucins were found to have similar amino acid profiles and to contain carbohydrate. It would appear therefore that a ‘link’ glycopeptide of molecular mass approx. 118 kDa is common to all of the gastrointestinal mucins studied.
An investigation was undertaken to discover whether mucin purified from secretions in the lumen of rat small intestine differed in structure or composition from intracellular mucin purified from rat intestinal tissue. To do this, ligated loops were constructed in situ from previously washed intestinal segments and mucin purified separately from tissue homogenates or loop fluid. Secreted mucin (SM) differed from intracellular mucin (IM) by having a higher proportion of ‘minor’ mucin amino acids (aspartic acid, glutamic acid, glycine and alanine) and a lower proportion of ‘major’ amino acids (serine, proline and threonine). SM also contained less N-acetylgalactosamine and a small, but measureable, amount of mannose. Gel electrophoresis showed that SM penetrated the gel more readily and, unlike IM, gave a rather prominent, but diffuse, band having a midpoint position of Mr 200,000. After reduction both IM and SM gave rise to the putative ‘link’ component of Mr 118,000 and the 200,000-Mr band of SM disappeared. SM was included to a greater extent than IM on Sepharose CL-2B chromatography, suggesting a smaller size. With the use of CsCl-density-gradient ultracentrifugation of SM, a lighter species [buoyant density (rho) = 1.38 g/ml] enriched in the 200,000-Mr component, was separated from a heavier, more glycosylated, species (rho = 1.50 g/ml). Purified 200,000-Mr component had a composition identical with that of the 118,000-Mr ‘link’ component of IM, reacted in Western blots with an antibody specific for the 118,000-Mr ‘link’ component, and after reduction gave rise to a 118,000-Mr component on gel electrophoresis. Thus secreted mucin contains a 200,000-Mr component which appears to represent a disulphide-linked dimer of the previously described 118,000-Mr ‘link’ component of intracellular mucin.
Rat intestinal mucin is polymerized by a putative ‘link’ component of Mr 118,000 that can be released from the native mucin by thiol reduction [Fahim, Forstner & Forstner (1983) Biochem. J. 209, 117-124]. To confirm that this component is an integral part of the mucin and independent of the mucin purification technique, rat mucin was purified in the present study by three independent techniques. In all cases, the 118,000-Mr component was released after reduction. The 118 kDa band was electroeluted from SDS/polyacrylamide gels and its composition shown to resemble closely that of the link component of human intestinal mucin [Mantle, Forstner & Forstner (1984) Biochem. J. 224, 345-354]. Carbohydrates were present, including significant (10 mol/100 mol) amounts of mannose, suggesting the presence of N-linked oligosaccharides. Monospecific antibodies prepared against the rat 118,000-Mr component established its tissue localization in intestinal goblet cells. Mucins subjected to SDS/polyacrylamide-gel electrophoresis and Western blots using the same antibody, established that the link components of rat and human intestinal mucin are similar antigenically. Brief exposure (10 min) of native rat mucin to trypsin or Pronase (enzyme/mucin protein, 1:500, w/w) also released a 118,000-Mr component that reacted with the monospecific antibody. Thus the 118,000-Mr component is an integral part of the mucin and, although linked to large glycopeptides by disulphide bonds, this component also has proteinase-sensitive peptide bonds, presumably at terminal locations such that brief treatment with proteinases releases the molecule in a reasonably intact form. Under physiological conditions, therefore, one might expect that, after mucin is secreted into the intestinal lumen, luminal proteinases would rapidly remove the link component, thereby causing the mucin to depolymerize.
Goblet-cell mucin of rat small intestine was purified from mucosal scrapings by using centrifugation, Sepharose 4B and Sepharose 2B chromatography. The mucin was applied in low concentrations (1 microgram/track) to slab gels containing 0.5% agarose/2% (w/v) polyacrylamide, and bands were detected after electrophoresis by silver stain or by fluorography of 3H-labelled mucin. Before reduction the mucin contained three distinct components: a polymeric species at the top of the gel and two large glycoproteins of higher mobility. After reduction, the polymer disappeared, the two glycoproteins remained unchanged, and two glycopeptide bands of higher mobility appeared. In addition, a non-glycosylated, heavily stained peptide of mol.wt. 118000 was detected. The individual mucin components were partially separated on Sepharose 2B, 0.2M-NaCl/1% sodium dodecyl sulphate being used as eluant. Individual amino acid and carbohydrate analyses suggested that the glycosylated components, despite their differences in size, had identical profiles. The 118000-mol.wt. peptide had a very different amino acid profile, with much less serine, threonine and proline. Glycine and aspartic and glutamic acids comprised 34% of the total amino acids. Thus the ‘native’ mucin is a heterogeneous structure containing at least two non-covalently associated glycoproteins plus polymeric material. The latter is stabilized by disulphide bonds and consists of several glycopeptides of different size as well as a ‘link’ peptide of mol.wt. 118000.