Biosynthesis of high molecular weight breast carcinoma associated mucin glycoproteins.
We have studied the biosynthesis of mucin glycoproteins recognized by monoclonal antibody W1 in MCF7 cells. Proteins of Mr 170,000, 185,000, 260,000, and 275,000 were immunoprecipitated from cells pulse-labeled with [3H]threonine and [3H]proline. Evidence suggesting that these proteins were precursors of high molecular weight mucin(s) included: 1) their kinetics of disappearance corresponded with appearance of mature mucin(s) (t1/2 = 30 min); 2) their processing into mature mucin(s) was not blocked by cycloheximide, but was disrupted by monensin, which impairs glycoprotein processing in the Golgi; 3) they were inaccessible to antibody added outside the cells, whereas mature mucin(s) was accessible and appeared at the cell surface with a t1/2 = 45 min; and 4) their mobilities of precursors varied between different cell lines, but generally correlated with mobilities of mature mucin(s). The precursors were sensitive to endoglycosidase H, indicating that they contained high mannose N-linked oligosaccharides. Less than 3% of threonine residues in the precursors, but more than 75% in mature mucin(s), were substituted with O-linked oligosaccharides. Therefore, initiation of N-linked oligosaccharides occurred soon after initiation of core protein synthesis, but initiation of O-linked oligosaccharides occurred much later, just prior to appearance of mature mucin(s) at the cell surface.
D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000378:metabolism*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D006207:Half-Life; D006801:Humans; D008970:Molecular Weight; D009077:Mucins / Q000096:biosynthesis*; D011392:Proline / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D009077:Mucins; D013912:Threonine; D011392:Proline