Biosynthesis of a human gall-bladder mucin.
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.
Klomp
L W
LW
Laboratory of Cell Biology, University of Utrecht, The Netherlands.
de Lely
A J
AJ
Strous
G J
GJ
D005260:Female; D005704:Gallbladder / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D009077:Mucins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D011498:Protein Precursors / Q000032:analysis / Q000378:metabolism
D009077:Mucins; D011498:Protein Precursors