Sulfation of the tumor cell surface sialomucin of the 13762 rat mammary adenocarcinoma.
ASGP-1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O-linked oligosaccharide chains. We have used this system to investigate the O-glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S-1 and S-2) were isolated as their oligosaccharitols by alkaline borohydride elimination, anion exchange HPLC, and ion-suppression HPLC. From structural analyses S-1 is proposed to be a branched, sulfated trisaccharide -O4S-GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and S-2 its sialylated derivative -O4S-GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP-1 intermediate in size between immature and mature sialomucin. Pulse-chase analyses showed that the intermediate could be chased into mature ASGP-1. The concomitant conversion of S-1 into S-2 had a half-time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and sialylated derivative GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNAc. These data suggest that sulfation of ASGP-1 is an intermediate synthetic step, which competes with beta-1,4-galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as beta-1,4-galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP-1.
Hull
S R
SR
Department of Anatomy and Cell Biology, University of Miami School of Medicine, Florida 33101.
Carraway
K L
KL
D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D009077:Mucins / Q000378:metabolism*; D009844:Oligosaccharides / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D052116:Sialomucins; D013464:Sulfuric Acids; D014407:Tumor Cells, Cultured / Q000378:metabolism
D009077:Mucins; D009844:Oligosaccharides; D052116:Sialomucins; D013464:Sulfuric Acids; D008985:Monensin