Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells

Abstract

Sialomucins are the dominant components of the cell surfaces of some carcinoma ascites cells and have been postulated to inhibit recognition of tumours by the immune system. The sialomucin ASGP-1 (ascites sialoglycoprotein-1) of the 13762 rat mammary adenocarcinoma is associated with the cell surface as a complex with a concanavalin-A-binding glycoprotein called ASGP-2. This sialomucin complex has been purified from ascites cell microvilli by extraction with Triton X-100 and CsCl density-gradient centrifugation. ASGP-1 (which has been purified previously) and ASGP-2 were dissociated in 6 M-guanidine hydrochloride and separated by gel filtration. The molecular mass of the undenatured detergent complex of ASGP-2, estimated by gel filtration and velocity sedimentation in Triton X-100, was 148 kDa. Since the apparent molecular mass by SDS/polyacrylamide-gel electrophoresis was about 120 kDa, ASGP-2 must be a monomer as extracted from the membrane. Studies of its chemical composition indicate that it contains about 45% carbohydrate by weight, including both mannose and galactosamine. Alkaline borohydride treatment of ASGP-2 converted approx. half of the N-acetylgalactosamine to N-acetylgalactosaminitol, demonstrating the presence of O-linked oligosaccharides. Analyses of mannose-labelled Pronase glycopeptides from ASGP-2 by lectin-affinity chromatography on concanavalin A and leucocyte-agglutinating phytohaemagglutinin suggested that 40% of the label was present in high-mannose/hybrid oligosaccharides, 20% in triantennary oligosaccharides substituted on the C-2 and C-4 mannose positions and 40% in tri- or tetra-antennary oligosaccharides substituted on C-2 and C-6. The presence of polylactosamine sequences on these oligosaccharides was suggested by lectin blots and by precipitation from detergent extracts with tomato lectin. From chemical analyses and lectin-affinity studies, we estimate that ASGP-2 contains four high-mannose and 13 complex N-glycosylated oligosaccharides, plus small amounts of polylactosamine and O-linked oligosaccharides. The presence of four different classes of oligosaccharides on this glycoprotein suggests that it will be an interesting model system for biosynthetic comparisons of the different glycosylation pathways.

Sialomucin complex at the rat ocular surface: a new model for ocular surface protection

“…We have described another membrane mucin, called sialomucin complex (SMC), which is composed of two subunits : an O-glycosylated mucin subunit ASGP-1 (ascites sialoglycoprotein-1) and an N-glycosylated integral membrane glycoprotein ASGP-2 [19,20]. The mucin subunit, ASGP-1, is tightly but noncovalently bound to the transmembrane subunit, ASGP-2.…”

Multiple facets of sialomucin complex/MUC4, a membrane mucin and ErbB2 ligand, in tumors and tissues (Y2K update)

“…The likely explanation is that a nonmembrane alternative splice form was sequenced in the human, though this question needs to be examined by both biochemical and molecular biological studies. Interestingly, both MUC1 and SMC/MUC4 are produced as heterodimeric complexes from a single gene product via a proteolytic cleavage during the early stages of their transit to the cell surface (32,33,34). Their cleavage sites appear to be unrelated (14).…”

Sialomucin Complex (Rat Muc4) Is Regulated by Transforming Growth Factor β in Mammary Gland by a Novel Post-translational Mechanism

“…SMC consists of a peripheral O-glycosylated mucin subunit ASGP-1 (7)(8)(9)(10) and an N-glycosylated integral membrane glycoprotein ASGP-2 (8,11). The complex is transcribed from a single gene as a 9-kilobase pair transcript (12,13) and translated into a single large polypeptide, which is proteolytically cleaved early in its biosynthesis.…”