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J Biol Chem. 1988 Mar 25;263(9):4215-22.

Biosynthesis of MAM-6, an epithelial sialomucin. Evidence for involvement of a rare proteolytic cleavage step in the endoplasmic reticulum.

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Division of Tumor Biology, The Netherlands Cancer Institute (Antoni van Leeuwenhoekhuis), Amsterdam.


MAM-6 is a major epithelial sialomucin which is abundantly present at the apical surface of ductal and alveolar cells of normal tissues and on many different carcinoma cells. MAM-6, as defined by monoclonal antibodies, consists of one or two sialylated glycoproteins with apparent molecular masses of over 400 kDa under reducing as well as nonreducing conditions. The mobility and number of glycoproteins immunoprecipitated vary depending on the cell line of origin. We have employed immunoprecipitation techniques to study the biosynthesis and glycosylation of this mucin. The biosynthesis of MAM-6 was studied in the ZR-75-1 breast cancer cell line. Two glycoproteins with apparent molecular masses of approximately 450 and 650 kDa, representing the mature form, were immunoprecipitated. By pulse-chase analysis, we show that the biosynthesis of the 450-kDa glycoprotein proceeded through intermediates of 220, 200, and 500 kDa which became perceptible after 1, 4, and 30 min, respectively. The biosynthesis of the 650-kDa glycoprotein followed a similar course through 380, 350, and 700-kDa intermediates. The processing of the 220- and 350-kDa precursors involves a rare proteolytic cleavage step which occurs in the endoplasmic reticulum. The late precursors of 500/700 kDa, observed after 30 min chase, and the mature glycoproteins were generated by extensive O-linked glycosylation. The formation of the 500/700-kDa precursors was not affected by monensin. However, the final step of maturation, sialylation of the 500/700-kDa precursors, could be inhibited by monensin. The extensive O-linked glycosylation causes the apparent high molecular weight as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data on the biosynthesis show that the early MAM-6 precursors contain N-linked glycans, suggesting the presence of N-linked glycans on the mature MAM-6 molecule. Although the copresence of N- and O-linked glycans has been found on many molecules, no N-linked glycans have been reported on mucus glycoproteins previously.

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