Biosynthesis of a tumor cell surface sialomucin. Maturation and effects of monensin

The major cell surface glycoprotein (ascites sialoglycoprotein-1 (ASGP-1] of ascites 13762 rat mammary tumor cells is a large (Mr greater than 500,000), highly glycosylated sialomucin which is present in great abundance (greater than 0.5% of total cell protein). Thus, these tumors provide a useful system for investigating the biosynthesis of O-glycosylated glycoproteins. Previous studies in this system have demonstrated that initiation of O-linked oligosaccharides occurs throughout most of the transit period of ASGP-1 from the endoplasmic reticulum to the cell surface. By pulse-chase threonine labeling and precipitation with peanut agglutinin, ASGP-1 is first observed as an immature lightly glycosylated form (Mr approximately 200,000) which is converted to a more mature, more heavily glycosylated form (designated the premature or P form) with a half-time of about 30 min. The P form is then more gradually converted into the mature ASGP-1. Analysis of glucosamine-labeled oligosaccharitols obtained from the immature form showed primarily unsialylated derivatives consisting of the structures of the size of the tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc and smaller, whereas the mature form showed a mixture of sialylated and unsialylated structures. Desialylation of glucosamine-labeled mature form resulted in a glycoprotein intermediate in size between the immature and mature forms, indicating that the size change with maturation is not solely due to sialylation. Treatment of the cells with 10(-6) M monensin significantly reduced the conversion of immature to mature form without inhibiting initiation of O-linked oligosaccharides and without preventing sialylation. Analysis of oligosaccharitols obtained from ASGP-1 of monensin-treated cells showed that the major oligosaccharides are trisaccharide GlcNAc beta 1,6(Gal beta 1,3)GalNAc and sialylated trisaccharide GlcNAc beta 1,6(NeuAc alpha 2,3-Gal-beta 1,3) GalNAc. These results suggest that monensin specifically disrupts the compartment of the biosynthetic pathway which adds most of the beta 1,4-Gal to the oligosaccharides of ASGP-1 and that this compartment is separate from the primary site of sialylation.     

Structural and functional aspects of tumor cell sialomucins

Sialomucins are abundant on the surfaces of certain ascites tumor cells and have been implicated in the escape of tumors from immune destruction and metastasis. They are large, highly glycosylated glycoproteins which are rich in serine and threonine and have a variety of 0-linked oligosaccharides. The sialomucin (ASGP-1) or 13762 rat mammary adenocarcinoma ascites cells represents more than 0.5% of the total cell protein and can be isolated from cell membranes by centrifugation in 4 M guanidine hydrochloride-cesium chloride. ASGP-1 can also be isolated from membranes or cells by nonionic detergent extraction as a 1:1 complex with a second glycoprotein ASGP-2. Studies with the fluorescent lectins peanut agglutinin, which binds ASGP-1, and Concanavalin A, which binds ASGP-2, indicate that the glycoproteins are present at the cell surface as a complex. ASGP-1 is shed into cell culture medium or ascites fluid, apparently by a proteolytic cleavage mechanism. 13762 ascites cells grown in culture or as solid tumors lose their ASGP-1. The sialomucin reappears with extensive passage of the tumor cells in ascites form. Studies on the biosynthesis of ASGP-1 indicate that carbohydrate is being added over nearly the entire period of transit of ASGP-1 from the site of polypeptide synthesis to the plasma membrane. The negatively charged, rod-like structure of the sialomucins suggests that they may play a role in inhibiting recognition or binding processes necessary for the immune destruction of these tumor cells.     

Temporal aspects of O-glycosylation and cell surface expression of ascites sialoglycoprotein-1, the major cell surface sialomucin of 13762 mammary ascites tumor cells

We have investigated the biosynthesis and cell surface expression of the major cell surface sialomucin (ascites sialoglycoprotein-1 (ASGP-1] of 13762 rat mammary ascites tumor cells by pulse or pulse-chase metabolic labeling combined with precipitation with peanut agglutinin and alkaline borohydride elimination or proteolytic fragmentation. The minimum time for initial glycosylation was estimated from the time required for the protein to acquire the ability to bind to peanut agglutinin to be less than 5 min. Moreover, when cells were labeled with threonine for 5 min and the ASGP-1 isolated by peanut agglutinin precipitation, 3% of the labeled threonine could be converted to 2-aminobutyric acid by alkaline borohydride elimination of the carbohydrate, indicating that at least 3% of the threonines of ASGP-1 are O-glycosylated within 5 min of polypeptide synthesis. The minimum time between the final glycosylation reactions in the cell and appearance of ASGP-1 at the cell surface was determined by trypsinizing galactose- or glucosamine-labeled cells at timed intervals after labeling to occur within 5-10 min of labeling. Both labeled glucosamine and galactosamine appeared in ASGP-1 fragments within 5 min, but the amount of labeled galactosamine was less than the amount of labeled glucosamine until after 20 min, when the 1:1 equilibrium ratio was reached. The half-time for appearance of glucosamine-labeled ASGP-1 at the cell surface was found to be greater than 4 h. The minimum time required from synthesis of the ASGP-1 polypeptide to appearance at the cell surface was determined by leucine labeling and proteolysis to be 70-80 min. These combined studies suggest a continuum of O-linked oligosaccharide initiation events extending over most of the period of ASGP-1 biosynthesis and transit from the endoplasmic reticulum to the cell surface.     

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

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.     

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.     

Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma : Relationship of the major sialoglycoprotein to xenotransplantability

The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-C1) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [³H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also labeled by lactoperoxidase and ¹²⁵I, indicating its presence at the cell surface. Metabolic labeling with [³H]glucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantable (MAT-C1) and non-xenotransplantable (MAT-B1) sublines, representing >70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labeling and transmission electron microscopy (TEM) do not show substantially greater amounts of negatively charged groups distributed along the cell surfaces of MAT-C1 than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.     

Biosynthesis of the cell surface sialomucin complex of ascites 13762 rat mammary adenocarcinoma cells from a high molecular weight precursor

Cell surfaces of metastatic 13762 ascites rat mammary adenocarcinoma cells are covered with a sialomucin complex composed of the high Mr sialomucin ASGP-1 (approximately 600,000) and a concanavalin A-binding, integral membrane glycoprotein ASGP-2 (120,000). Antibodies prepared against ASGP-2 and deglycosylated ASGP-1 react on immunoblots of ascites cells or their isolated microvilli with the Mr = 120,000 species and the high Mr sialomucin, respectively. No cross-reactivity was observed. Under complex dissociating conditions, anti-ASGP-2 immunoprecipitated primarily components of Mr = 120,000 and about 400,000 from lysates of cells labeled for 1 h with mannose, glucosamine, and threonine. Under similar conditions, anti-ASGP-1 immunoprecipitated the Mr = 400,000 component and a second major labeled component of about 330,000. Pulse-chase labeling with 35S-labeled amino acids followed by immunoprecipitation with anti-ASGP-2 indicated a precursor-product relationship for the Mr = 400,000 component, designated pSMC-1 (precursor, sialomucin complex), and ASGP-2. Similar pulse-chase analyses of threonine-labeled cells using anti-ASGP-1 showed equivalent amounts of immunoprecipitated pSMC-1 and pSMC-2, both of which disappeared with kinetics similar to those observed for pSMC-1 immunoprecipitated with anti-ASGP-2. A precursor-product relationship of both pSMC-1 and pSMC-2 to ASGP-1 was suggested by combined precipitations with anti-ASGP-1 and peanut agglutinin, which precipitates ASGP-1 specifically. Immunoblot and lectin blot analyses indicated that pSMC-1 and pSMC-2 from the immunoprecipitates bind anti-ASGP-2, anti-ASGP-1, and concanavalin A. Moreover, these three components can also be labeled with mannose; the mannose was removed from 30-min pulse-labeled anti-ASGP-2 immunoprecipitates by incubation with endo-beta-N-acetylglucosaminidase H, indicating the presence of only high mannose N-linked oligosaccharides in pSMC-1. One-dimensional peptide maps of 35S-labeled pSMC-1 and Mr = 120,000 ASGP-2 showed several corresponding bands. These results indicate that both ASGP-1 and ASGP-2 can be synthesized from a common high Mr precursor. We propose that complex is formed from pSMC-1 by proteolytic cleavage to yield Mr = 120,000 ASGP-2 plus the precursor to ASGP-1 early in the transit pathway from the endoplasmic reticulum to the cell surface.     

Evidence for the association of two cell surface glycoproteins of 13762 mammary ascites tumor cells : Concanavalin A-induced redistribution of peanut agglutinin-binding proteins

The behavior of the cell surface concanavalin A (conA) receptors and of peanut agglutinin (PNA) receptors on the MAT-B1 ascites subline of the 13762 rat mammary adenocarcinoma was examined using fluorescein-labeled conA and PNA. ASGP-1, the major glucosamine-containing glycoprotein of these ascites cells, is the only PNA-binding protein observed by dodecyl sulfate electrophoresis. ASGP-2, the second most prominent component after glucosamine labeling, is the most abundant conA-binding protein. These two glycoproteins were previously shown to be associated as a complex in detergent extracts of the cells [20]. ConA-binding proteins, upon incubation with fluorescein-labeled conA (FITC-conA), redistribute on the cell surface into small and large aggregates similar, but not identical, to those seen in ‘patching’ and ‘capping’ experiments with lymphocytes. PNA-binding proteins failed to redistribute during incubation with fluorescein-labeled PNA (FITC-PNA) and appeared in a diffusely stained pattern around the circumference of the cells. However, when cells were treated with unlabeled conA followed by FITC-PNA, or with FITC-PNA followed by unlabeled conA, there was marked redistribution of the FITC-PNA. These results indicate that ASGP-1 redistributes in response to the movement of conAbinding proteins and supports our hypothesis that ASGP-1 and ASGP-2 are associated on the plasma membrane at the cell surface as well as in detergent extracts.     

Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli

Membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the associated concanavalin A (Con A)-binding glycoprotein ASGP-2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP-1 from the microvilli and increases the association of ASGP-2 with the Triton-insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP-2 in the membrane and its association with the microfilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X-100 for the preparation of microfilament cores and Triton-soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L-phytohemagglutinin, and immunoblotting with anti-ASGP-2. The earliest major proteolysis product from this procedure was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa Triton-soluble fragments, and 25-30 kDa membrane- and microfilament-associated fragments were observed. Phalloidin shift analysis of microfilament-associated proteins on velocity sedimentation gradients indicated that the 25-30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP-2 has a site for indirect association with the microfilament core near the membrane on a 15-20 kDa segment.     

Mechanism of expression of Thomsen-Friedenreich (T) antigen at the cell surface of a mammary adenocarcinoma

The Thomsen-Friedenreich (T) antigen and its disaccharide component Gal beta 1,3GalNAc, which is recognized by the plant lectin peanut agglutinin (PNA), have been proposed as useful tumor markers because of their apparently specific occurrence in certain types of carcinomas. We have investigated the mechanism for the appearance of the disaccharide at the cell surface of ascites 13762 rat mammary adenocarcinoma cells using pulse-chase glucosamine labeling, proteolysis, and PNA precipitation of the cell-surface sialomucin ASGP-1. Glucosamine-labeled disaccharide appears at the cell surface in less than 10 min. Although the appearance of larger oligosaccharides continues to increase, the appearance of labeled disaccharide levels off within an hour. Analysis of intracellular vs. cell surface-labeled oligosaccharides showed that all disaccharide synthesized more than an hour before reaching the cell surface is converted to larger oligosaccharides. Thus, the presence of the disaccharide at the cell surface results from its synthesis late in the transit pathway of the sialomucin to the cell surface. We propose that the presence of T antigen at the surface of carcinoma cells results from an aberration of the pathway for O-linked glycosylation in these cells, probably caused by inappropriate localization of the enzymes involved in synthesis of the disaccharide.     

Changes in expression of a major sialoglycoprotein associated with ascites forms of a mammary adenocarcinoma

Glycoproteins of a cultured form (MR) of the 13762 rat mammary adenocarcinoma and its variants have been studied by analyses for peanut agglutinin receptors, [³H]glucosamine labeling, lactoperoxidase labeling and CsCl density gradient centrifugation. The 13762 MR cells, derived from 13762 MAT-B ascites cells, do not contain detectable ASGP-1, the predominant cell surface sialoglycoprotein of the ascites forms of the 13762 tumor.Transplantation and continued passage as ascites cells of MR cells or clonal lines derived from MR results in abrupt expression of ASGP-1 at about passage 16; it is absent in early passages of the ascites tumor. When these ascites cells are transferred to culture, ASGP-1 is again lost. No ASGP-1 is found in solid tumors derived from subcutaneous transplantation of the 13762 MR cells. The results suggest modulation of ASGP-1 content of the 13762 tumor cells.     

Identification and characterization of a novel antigen complex on mouse mammary tumor cells using a monoclonal antibody against platelet glycoprotein Ic

The rat monoclonal antibody GoH3 identifies a complex of glycoproteins Ic and IIa on human and mouse platelets. The GoH3 epitope is located on glycoprotein Ic. A novel glycoprotein complex is identified by GoH3 on the surface membranes of mouse mammary epithelial tumor cells. This antigen complex is composed of glycoprotein Ic noncovalently associated with a monomor or a disulfide-linked multimer of a high molecular weight glycoprotein (Ic-binding protein (IcBP]. Glycoprotein Ic is synthesized as a large precursor with asparagine N-linked high mannose oligosaccharides. Processing of this precursor involves a proteolytic cleavage of the large polypeptides into two smaller disulfide-linked polypeptide chains, Ic alpha (heavy) and Ic beta (light), and conversion of the majority of the high mannose oligosaccharides into complex-type glycans. Likewise, glycoprotein IcBP is initially glycosylated with high mannose asparagine N-linked oligosaccharides which are processed to complex units in the mature form. Association of glycoprotein Ic with IcBP occurs within the cell soon after their synthesis. The kinetics of labeling show non-coordinate processing consistent with the idea that the concentration of glycoprotein Ic limits complex formation and the subsequent processing of glycoprotein IcBP.     

Shedding of the major plasma membrane sialoglycoprotein from the surface of 13762 rat ascites mammary adenocarcinoma cells

ASGP-1 (ascites Sialoglycoprotein 1) the major sialoglycoprotein of 13762 rat ascites mammary adenocarcinoma cells, is shed from MAT-B1 (nonxenotransplantable) and MAT-C1 (xenotransplantable) sublines when incubated in vitro after labeling in vivo with [³H]glucosamine. The rates of shedding of label in both particulate and soluble form are similar for the two sublines, but the turnover of label in the cells is 80% greater for MAT-C1 cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 2.4 days) than for MAT-B1 cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 4.1 days). Shed soluble ASGP-1 was smaller than ASGP-1 in the particulate fraction by gel filtration in dodecyl sulfate. By CsCl density gradient centrifugation, gel filtration, and sucrose density gradient centrifugation, all in 4 m guanidine hydrochloride, the shed soluble ASGP-1 was found to be slightly more dense and smaller than ASGP-1 purified from membranes. No differences in sialic acid or oligosaccharides released by alkaline borohydride treatment were found between the shed soluble ASGP-1 and purified ASGP-1. These results suggest that the shed soluble ASGP-1 is released from the membrane by a proteolytic cleavage. This mechanism is supported by the inhibition of the release of soluble shed ASGP-1 by aprotinin, a protease inhibitor. Soluble ASGP-1 in ascites fluid is also smaller by gel filtration, but is more heterogeneous, suggesting a similar release mechanism in vivo followed by more extensive degradation in the ascites fluid.     

Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.     

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.     

N-Acetylneuraminic acid and N-glycolylneuraminic acid in the O-linked oligosaccharides of a tumor cell glycoprotein. Incorporation and distribution

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, which differ in several cell surface properties, contain a major mucin-type glycoprotein, termed ASGP-1. The sialic acid content of MAT-C1 ASGP-1 is 2-3-fold greater than MAT-B1 ASGP-1 (Sherblom, A. P., Buck, R. L., and Carraway, K. L. (1980) J. Biol. Chem. 255, 783-790). Sialic acid analysis demonstrated that, whereas MAT-C1 ASGP-1 contained approximately equal amounts of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl), MAT-B1 ASGP-1 was devoid of NeuGl. MAT-B1 microsomes also did not contain NeuGl. MAT-B1 cells incubated with [3H]N-acetylmannosamine did not synthesize either labeled CMP-NeuGl or free NeuGl, even though the CMP-sialic acid synthetase was active with the substrate NeuGl. Thus, MAT-B1 cells may be deficient in the enzyme N-acetylneuraminate monooxygenase. The O-linked oligosaccharides from both MAT-B1 and MAT-C1 ASGP-1 have been shown to contain a core tetrasaccharide Gal(beta 1-4)GlcNAc(beta 1-6)(Gal(beta 1-3]GalNAc in which both galactose residues may be linked to additional sugars (Hull, S. R., Laine, R. A., Kaizu, T., Rodriquez, I., and Carraway, K. L. (1984) J. Biol. Chem. 259, 4866-4877). The distribution of NeuAc and NeuGl between the two galactose termini of the core tetrasaccharide was examined for MAT-C1 ASGP-1. Oligosaccharides were released by alkaline-borohydride treatment of MAT-C1 ASGP-1 which had been labeled with [14C]glucosamine and galactose oxidase/B3H4. Following fractionation by Bio-Gel P-4, DEAE-Sephadex, and high-performance liquid chromatography, oligosaccharides were analyzed for NeuAc and NeuGl and for susceptibility to digestion with beta-galactosidase. Three disialylated oligosaccharides were identified containing 2 mol of NeuAc (5.5% recovery), 2 mol of NeuGl (4.5%), or 1 mol each of NeuAc and NeuGl (11.1%). For monosialylated oligosaccharides, NeuGl appeared preferentially associated with the Gal(beta 1-4)GlcNAc terminus (9.0%), whereas significant amounts of oligosaccharide containing NeuAc at both the Gal(beta 1-3)GalNAc (2.6%) and Gal(beta 1-4)GlcNAc (4.5%) termini were detected. Each of the major qualitative differences between MAT-B1 and MAT-C1 oligosaccharides, including the presence of NeuGl (MAT-C1), sulfate (MAT-B1), and alpha-linked galactose (MAT-B1), occurs at the Gal(beta 1-4)GlcNAc terminus.     

Structures of the O-linked oligosaccharides of the major cell surface sialoglycoprotein of MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma

Structures of the principal O-glycosides from the major cell surface sialoglycoprotein (ASGP-1) of the MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma have been determined. Oligosaccharitols were released by alkaline borohydride treatments of ASGP-1 and purified by gel filtration, DEAE-Sephadex ion exchange chromatography, and high performance liquid chromatography. On the basis of carbohydrate composition, methylation analysis, periodate oxidation, and exoglycosidase digestion, the five major oligosaccharides released by mild alkaline borohydride were assigned the following structures: Component II-3: (NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)Ga 1 NAcOH(3----1 betaGa 1 3----2 alpha NeuAc) III-2a: (Ga 1 beta 1----4G1cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) III-2c: (Ga 1 alpha 1----3Ga 1 beta 1----4G1cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) IV-1a: (Ga 1 beta 1----4G 1 cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1) IV-1c: (Ga 1 alpha 1----3Ga 1 beta 1----4G 1 cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1) Fucosylated derivatives of III-2a, IV-1a, and IV-1c were found in smaller amounts with the fucose tentatively assigned to the 2-position of the lactosamine galactose. Components II-3, III-2a, and the fucosylated derivative of III-2A were found in both MAT-B1 and MAT-C1 sublines. The alpha-galactosides were found in detectable quantities only in subline MAT-B1. Oligosaccharides from MAT-C1 cells were enriched in sialic acid when compared to those from MAT-B1 cells. These results suggest that the 13762 ascites sublines, which bear different oligosaccharides, will provide models useful for the investigation of mechanisms regulating the expression of structures of the larger O-linked oligosaccharides.     

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.     

Isolation and characterization of a human T lymphocyte-associated glycoprotein (gp40)

The biosynthetic and structural characteristics of the human thymocyte/T cell antigen defined by the monoclonal antibody WT1 have been studied. WT1 identified a monomeric cell surface glycoprotein of Mr = 40,000 ( gp40 ). Cross-absorption experiments and two-dimensional gel analyses indicate that WT1 and another monoclonal antibody, 3A1, react with the same structure. This glycoprotein was asymmetrically inserted into the rough endoplasmic reticulum as a transmembrane structure. At this stage, the polypeptide chain possessed two N-linked, "high-mannose" type glycans; these were subsequently processed into endo-H-insensitive, complex oligosaccharides during intracellular transport to the cell surface. Inhibition of N-linked glycosylation with tunicamycin failed to block the processing of the nonglycosylated Mr = 29,000 polypeptide to a glycoprotein of Mr = 33,000. Cleavage of the mature Mr = 40,000 form with endo-F yielded a similar Mr = 33,000 product. The kinetics of synthesis of the Mr = 33,000 intermediate in conjunction with gal-NAc oligosaccharidase digestion indicated the presence of O-linked glycans in the mature cell surface WT1 antigen. The fully processed cell surface form of the polypeptide also contains covalently associated fatty acid, and was labeled by 32P phosphate, the predominantly labeled phosphoamino acid being phosphoserine. We also demonstrate biochemically that the reactivity of WT1 with cells from a few patients with acute myeloid leukemia reflects genuine expression of the gp40 structure on myeloid cells.