Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization.
Gastric mucin plays an important role in the protection of the stomach wall from chemical, microbiological and mechanical damage. We have previously isolated human gastric mucus glycoproteins and raised a polyclonal antiserum against these macromolecules. This antiserum specifically reacted with gastric mucins in immunoblotting experiments and stained mucous granules at the apical side of gastric surface epithelial cells. A similar staining pattern was obtained after incubation with an antiserum against rat gastric mucin. Next we used the antiserum in pulse-chase experiments of human stomach tissue explants. After short labelling periods with [35S]methionine and [35S]cysteine, the antiserum reacted with a polypeptide with an apparent molecular mass of approx. 500 kDa as determined by SDS/PAGE, which was converted after 90 min into a heterogeneous high-molecular-mass glycoprotein. This high-molecular-mass form, but not the 500 kDa polypeptide, was detectable in the culture medium after 2 h. This strongly suggests that the 500 kDa polypeptide is the precursor of the purified gastric mucin. Analysis of pulse-chase experiments by non-reducing SDS/PAGE revealed that the precursors form disulphide-linked oligomers early in biosynthesis, before the addition of O-linked sugars. After preincubation with the N-glycosylation inhibitor, tunicamycin, the apparent molecular mass of the precursor decreased marginally but consistently, indicating that N-linked glycan chains are present on the mucin precursor.
Klomp
L W
LW
Laboratory of Cell Biology, University of Utrecht, The Netherlands.
van Rens
L
L
Strous
G J
GJ
D005260:Female; D005748:Gastric Fundus / Q000737:chemistry / Q000378:metabolism; D005752:Gastric Mucins / Q000096:biosynthesis*; D005753:Gastric Mucosa / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007120:Immunochemistry; D008297:Male; D011233:Precipitin Tests; D011498:Protein Precursors / Q000032:analysis / Q000378:metabolism*; D013270:Stomach / Q000737:chemistry
D005752:Gastric Mucins; D011498:Protein Precursors