Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue.

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.     

Rapid intramolecular turnover of N-linked glycans in plasma membrane glycoproteins. Extension of intramolecular turnover to the core sugars in plasma membrane glycoproteins of hepatoma

Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl Acad. Sci. USA 80, 4026-4029]. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. Intramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with Mr of 85,000 (hgp85), 105,000 (hgp105), 115,000 (hgp115), 125,000 (hgp125), 175,000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin-A--Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgp105, hgp115 and hgp125 revealed the presence of N-linked oligosaccharides containing L-fucose, D-galactose, D-mannose and N-acetyl-D-glucosamine, but only of trace amounts of N-acetyl-D-galactosamine; hgp175 additionally contained significant amounts of N-acetyl-D-galactosamine, indicating the presence of both N- and O-linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N-linked oligosaccharides of hgp105, hgp115, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosaccharides of the high-mannose type. Half-lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse-chase experiments in vivo, using L-[3H]fucose as a marker of terminal sugars, D-[3H]mannose as marker of a core sugar and L-[3H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half-lives ranging over 41-90 h with a mean of 66 h. Compared to the degradation of the polypeptide backbone, both the terminal sugar L-fucose and the core sugar D-mannose turned over with much shorter half-lives averaging about 20 h in the five glycoproteins. The data show that, conversely to liver, within plasma membrane glycoproteins of hepatoma not only peripheral sugars but also core sugars of the oligosaccharides are split off during the life-span of the protein backbone. It may therefore be assumed that this reprocessing of plasma membrane glycoproteins is sensitive to malignant transformation.     

Comparative study on fucosylation of chicken liver and virus-induced hepatoma Mc-29

Comparative studies on fucoprotein metabolism of chicken liver and hepatoma Mc-29 have been carried out and the following parameters were determined: the incorporation rate of [14C]fucose into hepatoma and liver total tissue homogenate, acid-soluble and acid-insoluble fractions, acid-soluble nucleotide fraction and into plasma-membrane acid-precipitable fraction; the activity of microsomal and plasma-membrane fucosyltransferase; the electrophoretic pattern of hepatoma and liver plasma-membrane proteins and the incorporation of [14C]fucose into the glycoprotein fractions in both plasma-membrane preparations. It was found that the labelling of hepatoma tissue homogenate and plasma membranes was higher than that of the same liver preparations 3 hr after the [14C]fucose injection. This finding was supported by a considerably elevated hepatoma fucosyltransferase activity. The labelling rate of numerous fucoproteins from hepatoma plasma membranes was greatly increased and some of the individual glycoprotein bands were labelled to a higher extent compared with liver. The data presented show specific alterations of fucose and fucoprotein metabolism which could be considered as a characteristic feature of chicken viral-induced hepatoma Mc-29.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

Gangliosides of plasma membranes from normal rat liver and Morris hepatoma.

Plasma membranes were isolated from normal rat liver and Morris hepatoma 5123tc by discontinuous sucrose gradient centrifugation. There was an average two and one-halffold enrichment of gangliosides in plasma membranes from normal liver and hepatoma as compared with their respective whole cells. The amount of total gangliosides in plasma membranes from hepatoma was eight times greater than that found in normal liver. This increase resulted from a fivefold increase in hematosides, an eightfold increase in monosialogangliosides and a twenty-twofold increase in disialogangliosides. Trisialogangliosides were present in normal liver but not in hepatoma.     

Decreased intramolecular turnover of L-fucose in membrane glycoproteins of rat liver during liver regeneration

In plasma membrane glycoproteins of rat liver L-fucose undergoes a rapid intramolecular turnover in that fucose residues are removed from the glycoproteins (Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4026-4029). The present paper demonstrates that the intramolecular turnover of L-fucose is markedly decreased during liver regeneration. Turnover half-lives of L-fucose were measured in regenerating liver by pulse-chase experiments in five plasma membrane glycoproteins (Mr 60,000 (gp60), 80,000 (gp80), 120,000 (gp120), 140,000 (gp140), and 160,000 (gp160). The glycoproteins were isolated from plasma membranes by concanavalin A-Sepharose affinity chromatography and semipreparative NaDodSO4 polyacrylamide gel electrophoresis. L-Fucose turned over in the five glycoproteins with heterogeneous half-lives ranging from 22 h (gp160) to 49 h (gp120). The protein moieties of the glycoproteins were degraded with half-lives ranging from 56 h (gp80) to 107 h (gp140). Relative to the half-life of the protein backbone the half-live of L-fucose was increased in the five membrane glycoproteins by 70% (gp60), 150% (gp80), 182% (gp120), 60% (gp140) and 16% (gp160) during liver regeneration when compared to normal liver. The data show that L-fucose turns over in different membrane glycoproteins with individual rates, and that loss of L-fucose from plasma membrane glycoproteins is reduced in rapidly proliferating liver after partial hepatectomy.     

Studies on regenerating liver and hepatoma plasma membranes--II. Membrane fluidity and enzyme activity

1. Rat hepatocyte plasma membranes isolated from Morris hepatoma 7288C, normal and regenerating liver were labelled with the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. 2. Steady-state fluorescence polarisation measurements indicated an increased fluidity of the membranes in the early stages of regeneration, returning to normal levels after 48 hr. 3. There was a decrease in hepatoma plasma membrane fluidity compared to normal hepatocytes. Changes in fluorescence polarisation with temperature (Arrhenius studies) indicate an increase in the lower critical temperature for the membrane lipid thermotropic transition of hepatoma compared to normal liver plasma membranes. 4. These changes in membrane lipid fluidity alter the activation of some intrinsic and extrinsic membrane bound enzymes.     

Integral membrane antigens involved in cell-substratum adhesion of hepatocytes and hepatoma cells

Cell-substratum adhesion of rat hepatocytes was inhibited by antisera raised against purified plasma membranes of rat liver (anti-liver-antiserum) and Morris hepatoma 7777 (anti-hepatoma-antiserum). It is assumed that substances which block the adhesion-inhibiting activity of the antisera are involved in cell-substratum adhesion. Adhesion-involved molecules of rat liver monitored as 'blocking activity' were compared with those of Morris hepatoma 7777 and 9121. They were found to be integral membrane glycoproteins, which could be solubilized only by detergents. Fractionation of plasma membrane extracts by size exclusion HPLC revealed two blocking activity peaks representing molecules involved in the adhesion to plastic (P-AIM) and collagen (C-AIM). In rat liver both adhesion-involved molecules were found; yet P-AIM seemed to be the major type of adhesion-involved molecule. In the relatively well differentiated Morris hepatoma 9121 also both types were detected. In membrane extracts of the high malignant and poorly differentiated Morris hepatoma 7777, however, no P-AIM but only C-AIM were found. Estimation by size exclusion HPLC revealed molecular weights of 120 kD for C-AIM and approx. 105 kD for P-AIM. On SDS gel electrophoresis proteins in the region of 95 kD were found in C-AIM containing fractions, whereas proteins of 105 kD are likely candidates for P-AIM.     

Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes.

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.     

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.     

Increased expression of a high molecular weight matrix component in rat hepatocellular carcinoma

The urea extract of the glycoproteins from the extracellular matrix of rat liver has been compared with that of Morris hepatoma 7777. A high molecular weight glycoprotein present in Morris hepatoma 7777 was not found in the extract of liver matrix. Under reducing conditions in SDS-gel electrophoresis this component gave two glycoprotein bands with Mr 53 k and 56 k. The indirect immunofluorescence staining with a monospecific antiserum directed against the component showed its abundant presence in Morris hepatoma 7777 as well as in the less malignant Morris hepatoma 9121 in form of extracellular network structures. The antigen also densely filled some cumuli of cells. In contrast the liver tissue showed only very weak staining of the extracellular areas. The overall distribution of the component could be correlated with the distribution of several hydrolases in the tumor matrix, notably beta-D-glucuronidase.     

Differential response of the NADH oxidase of plasma membranes of rat liver and hepatoma and HeLa cells to thiol reagents

NADH oxidase activity of plasma membranes from rat hepatoma and HeLa cells responded to thiol reagents in a manner different from that of plasma membranes of liver. Specifically, the NADH oxidase activity of plasma membranes of HeLa cells was inhibited by submicromolar concentrations of the thiol reagentsp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), or 5,5-dithiobis-(2-nitrophenylbenzoic acid) (DTNB), whereas that of the rat liver plasma membranes was unaffected or stimulated over a wide range of concentrations extending into the millimolar range. With some hepatoma preparations, the NADH oxidase activity of hepatoma plasma membranes was stimulated rather than inhibited by PCMB, whereas with all preparations of hepatoma plasma membranes, NEM and DTNB stimulated the activity. In contrast, NADH oxidase activity of rat liver plasma membrane was largely unaffected over the same range of PCMB concentrations that either stimulated or inhibited with rat hepatoma or HeLa cell plasma membranes. Dithiothreitol and glutathione stimulated NADH oxidase activity of plasma membranes of rat liver and hepatoma but inhibited that of HeLa plasma membranes. The findings demonstrate a difference between the NADH oxidase activity of normal rat liver plasma membranes of rat hepatoma and HeLa cell plasma membranes in addition to the differential response to growth factors and hormones reported previously (Brunoet al., 1992). Results are consistent with a structural modification of a NADH oxidase activity involving thiol groups present in plasma membranes of rat hepatoma and HeLa cells but absent or inaccessible with plasma membranes of rat liver.     

Studies on regenerating liver and hepatoma plasma membranes--I. Lipid and protein composition

1. Plasma membranes were isolated from normal liver, Morris hepatoma 7288C and regenerating liver, 6, 15, 24, and 48 hr after partial hepatectomy. 2. The cholesterol/phospholipid ratio was lower in regenerating liver 6 hr after partial hepatectomy (0.51) compared to the sham control (0.68), returning to normal after 15 hr. This was accompanied by a small increase in palmitic acid (16:0). There were no other changes in the lipid composition in regenerating hepatocytes in the first 48 hr after partial hepatectomy. 3. Analysis of lipid composition showed a higher cholesterol/phospholipid ratio in the hepatoma plasma membrane compared to normal liver accompanied by an increase in saturation of the fatty acyl groups of the phospholipids. There were also significant changes in the phospholipid classes. 4. There was no change in the two-dimensional electrophoretic profile of membrane proteins in the early stages of liver regeneration, however hepatoma membranes showed significant differences in protein profile. 5. These changes in the lipid composition of the hepatoma plasma membrane would have the effect of decreasing the average fluidity of the membrane and together with the changes in protein composition may be significant in the altered growth of the hepatoma. Changes in the lipid composition of the hepatocyte plasma membrane early in liver regeneration may reflect the onset of renewed cell division.     

Effects of the Amnesic Agent 2-Deoxygalactose on Incorporation of Fucose into Chick Brain Glycoproteins

The interaction of the amnesic agent 2-deoxygalactose with fucose incorporation into glycoproteins in day-old chick forebrain has been studied with the aim of identifying glycoproteins whose synthesis is modified during memory formation. 2-Deoxygalactose inhibited total exogenous [14C]fucose incorporation into the forebrain glycoproteins by 26%. Sodium dodecyl sulphate-polyacrylamide gradient gel analysis revealed that intracerebrally injected 2-[3H]deoxygalactose labelled the same eight major glycoprotein bands as were identified using [14C]fucose labelling. Subsequent investigations focussed on these selected components. Subcellular fractionation showed that between 4 and 24 h after administration of the deoxy-sugar, the incorporated radioactivity was found predominantly at the synaptic sites, some glycoproteins being more abundant in synaptic plasma membranes and others in postsynaptic densities. This distribution pattern varied according to the time after injection. The effect of passive avoidance training, using a methylanthranilate-coated bead, on [14C]fucose incorporation into forebrain was to decrease fucose uptake into components of molecular mass 150-180 kilodaltons but to increase significantly labelling of glycoproteins of molecular mass 33 and 28 kilodaltons. The possible implications of these training-induced changes are discussed.     

Rates of degradation of glycoproteins from normal and regenerating rat livers: A study using double isotopes

The average decay rates (half-lives) of mixed glycoproteins were measured using double isotopes of fucose and glucosamine and compared to those of mixed overall proteins measured with leucine and NaH¹⁴CO₃ in whole homogenates and plasma membranes from normal and regenerating rat livers. A large reutilization of leucine was observed under both normal and regenerating conditions. Fucose seems to be recycling most predominantly in regenerating liver, whereas glucosamine was found to be very little if not at all reutilized under both conditions. Comparison of the results obtained with NaH¹⁴CO₃ and glucosamine demonstrated that glycoproteins from normal liver homogenate are degraded at a faster rate than mixed proteins. Contrary to that of mixed proteins, the half-life of glycoproteins remains unchanged during liver regeneration, and the use of glucosamine revealed that the degradation of plasma membrane glycoproteins is identical to that found in whole homogenate under both normal and regenerating conditions. Finally, the relative degradation rates of fractionated plasma membrane proteins and glycoproteins were evaluated under the same conditions. During liver regeneration some readjustments are observed in the relative degradation rates of individual species which suggest that the synthesis and degradation of the various surface membrane glycoproteins proceed at rates that are controlled independently.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Purification of liver and hepatoma membrane proteins by high-performance liquid chromatography

This paper documents the recovery of selected proteins from hepatic plasma membranes. Initial purification, achieved by a series of stepwise extractions, facilitates the subsequent purification by HPLC. Examples are provided to illustrate the recovery of specific proteins from two Morris hepatoma lines and the liver.     

Lipid peroxidation and fluidity of plasma membranes from rat liver and Morris hepatoma 3924A

Plasma membranes isolated from the fast-growing, maximal-deviation, Morris hepatoma 3924A exhibit remarkable changes in lipid composition, lipid peroxidation and to some extent in the physical state with respect to rat liver plasmalemmas. A correlation appears to exit between the lower phospholipid: protein ratio, higher cholesterol: phospholipid ratio, lower rate of lipid peroxidation and decrease in fluidity in tumor plasma membranes.     

Comparative proteomics of rat liver and Morris hepatoma 7777 plasma membranes

Plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized by use of different reagents. After selective solubilization, proteins were identified by nano-HPLC-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Using simple software, the patterns of proteins identified in membrane solubilizates from liver and hepatoma were compared. Proteins identified in Morris hepatoma 7777 and not in the corresponding membrane solubilizate from liver, mostly members of the annexin and heat shock protein families, are discussed as potential candidate markers for hepatocellular carcinomas.     

Increased expression of a high molecular weight matrix component in rat hepatocellular carcinoma

Summary The urea extract of the glycoproteins from the extracellular matrix of rat liver has been compared with that of Morris hepatoma 7777. A high molecular weight glycoprotein present in Morris hepatoma 7777 was not found in the extract of liver matrix. Under reducing conditions in SDS-gel electrophoresis this component gave two glycoprotein bands with M_r 53 k and 56 k. The indirect immunofluorescence staining with a monospecific antiserum directed against the component showed its abundant presence in Morris hepatoma 7777 as well as in the less malignant Morris hepatoma 9121 in form of extracellular network structures. The antigen also densely filled some cumuli of cells. In contrast the liver tissue showed only very weak staining of the extracellular areas. The overall distribution of the component could be correlated with the distribution of several hydrolases in the tumor matrix, notably -d-glucuronidase.Dedicated to Professor G.-W. Löhr on the occasion of his 65th birthdaySupported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, H.H.R. is recipient of grant Ri 421/1-1 from the Deutsche Forschungsgemeinschaft