Sulfation of the tumor cell surface sialomucin of the 13762 rat mammary adenocarcinoma

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Abstract
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 boro-hydride elimination, anion exchange HPLC, and ion-suppression HPLC. From structural analyses S-1 is proposed to be a branched, sulfated trisaccharide −O4S-GlcNAcβ1,6-(Galβ1,3)-GalNAc and S-2 its sialylated derivative −O4S-GlcNAcβ1,6-(NeuAcα2,3-Galβ1,3)-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 GlcNAcβ1,6-(Galβ1,3)-GalNAc and sialylated derivative GlcNAcβ1,6-(NeuAcα2,3-Galβ1,3)-GalNac. These data suggest that sulfation of ASGP-1 is an intermediate synthetic step, which competes with β-1,4-galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as β-1,4-galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP-1.