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Biosynthesis of a human gall-bladder mucin.

ABSTRACT: Mucin glycoproteins play an important role in the initial stages of gall-stone formation by a currently largely unknown mechanism. Understanding the structure of gall-bladder mucin is necessary to comprehend the mechanism by which cholesterol monohydrate crystals aggregate. Three successive CsCl-gradient-ultracentrifugation steps were used to purify human gall-bladder mucin from gall-bladder tissue. The isolated macromolecules had a typical mucin-like monosaccharide composition and appeared as heterogeneous high-M(r) glycoproteins on SDS/PAGE. A polyclonal antiserum was raised against these molecules and the specificity of the antiserum was ascertained by immunoblotting. The antiserum specifically stained mucous granules at the apical side of all gall-bladder epithelial cells in neck, fundus and body. The antibody was subsequently used to immunoprecipitate the mucin and biosynthetic intermediates from gall-bladder-tissue homogenates. An early biosynthetic precursor of the isolated mucin was identified by SDS/PAGE as a single polypeptide with an apparent M(r) of approx. 470,000. This precursor protein was converted after 1 h into a heterogeneous high-M(r) glycoconjugate with an electrophoretic mobility similar to that of the purified mucin. The mature mucin, but not the precursor, was secreted into the culture medium, starting at 1 h. As shown by SDS/PAGE under non-reducing conditions, the precursors form disulphide-linked oligomers. Using the N-glycosylation inhibitor tunicamycin, the apparent M(r) of the precursor was decreased to approx. 410,000, indicating that N-linked glycan chains are attached to the precursor polypeptide.

SUBMITTER: Klomp LW

PROVIDER: S-EPMC1137396 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Project description:The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level.Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses.We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins.We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function.

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Biosynthesis of a human gall-bladder mucin.

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