Lymphocyte plasma membranes. VI. Plasma membrane glycoproteins of thymic and splenic lymphocytes from inbred rats.

Plasma membranes of splenic and thymic lymphocytes from ACI rats were analyzed for their protein and glycoprotein components by surface radioiodination with 125I and SDS-polyacrylamide gel electrophoresis. The glycoproteins were extracted with lithium diiodosalicylate, characterized and assayed with antisera to thymic antigen. Plasma membranes of both cell types showed more than 25 proteins of which 10--15 were glycoproteins. Both cells showed five major glycoproteins but their apparent molecular weights or intensities differed. Surface radioiodination showed a 120 000 daltons component, common to both cell types, and a 27 000 daltons thymus-specific component as the most exposed surface glycoproteins. Lithium diiodosalicylate extracts of the plasma membranes contained almost all of the glycoprotein components and comprised 5-6 percent of the total membrane protein and 40-50 percent of the total membrane carbohydrate, with sialic acid content in thymus twice that of the spleen cells. About 1 percent of the total plasma membrane protein and 7 percent of the total isolated glycoproteins from thymocytes were reactive with rabbit anti-rat thymocyte antiserum and the immune precipitates showed two components with apparent molecular weights of 72 000 and 27 000.     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.     

Lipids and caplain in guinea pig tissues in the process of development of experimental autoimmune encephalomyelitis

The character of some lipids level change--cholesterol and phospholipids--as basic lipid components of cell membranes in the guinea-pig brain and liver tissue, and in serum in conditions of development of experimental autoimmune encephalomyelitis (EAE) have been investigated on the 11th, 21st, 27th day after inoculation. It has been detected, that the level of the investigated lipids changes wavely and indifferent-direction in the brain tissue on the 21st day of EAE. Similar variability observed in the activity of proteolytic ferment calpain, which is authentically reduced in the brain tissue by the 11th hour and increases up to the test objective level in the subsequent periods of EAE development. In the liver the level of alpha-tocopherol is reduced, while the content of studied lipids does not change. The investigated parameters can be attributed to the factors, which play an essential role in structural stability of cell membranes and their variability in conditions of EAE development is related to the processes of nervous cells demyelinisation and, hence to occurrence of such pathology as multiple sclerosis in people.     

A convenient procedure for producing gram-quantities of brucine l-guluronate and brucine d-mannuronate

A comparative investigation of the individual protein-bound carbohydrate components and the distribution of carbohydrates in the electrophoretic glycoprotein fractions was carried out in sera from 20 normal subjects and 30 schizophrenic patients matched for age and sex. The mean concentration of each of the protein-bound carbohydrate components was significantly elevated in schizophrenics. The electrophoretic patterns for serum glycoprotein showed increases in alpha-2 and beta globulins in schizophrenics. The serum glycoproteins contained glucose and l-arabinose, in addition to mannose, galactose, fucose, sialic acid, and a trace of xylose. The identity of glucose and arabinose was confirmed by g.l.c.-electron-impact mass spectrometry and by specific enzymic reactions. The contents of glucose and arabinose were higher in serum glycoproteins from schizophrenic patients. This elevation of serum glycoprotein paralleled serum glycosaminoglycan elevation previously reported by us, but was opposite to decrease of urinary glycoprotein in schizophrenics.     

Tumour-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.     

CHARACTERIZATION AND TOPOGRAPHY OF THE GLYCOPROTEINS OF ADRENAL CHROMAFFIN GRANULES

The glycoproteins of the membranes of bovine chromaffin granules were characterized by two polyacrylamide gel electrophoresis systems. Five components (I-V) were demonstrated with apparent molecular weights ranging in the unreduced form from 45,000 to 150,000. Glycoprotein I was identified as the enzyme dopamine β-hydroxylase. Four of these glycoproteins (with the exception of component IV) were apparently also present in the membranes of pig and horse chromaffin granules. The soluble proteins of chromaffin granules contained at least three glycoproteins. Only glycoprotein I (dopamine β-hydroxylase) was present both in the soluble content and in the membranes of chromaffin granules. Affinity chromatography with lectins demonstrated that from the soluble proteins only dopamine β-hydroxylase was adsorbed by concanavalin A, whereas none of these proteins reacted with wheat germ lectin and Ricinus communis agglutinin. Three membrane proteins including dopamine β-hydroxylase and glycoprotein II as major components were adsorbed by concanavalin A, whereas wheat germ lectin bound only component II and a small amount of component III. By electron microscopy it was demonstrated that concanavalin A did not bind to intact chromaffin granules whereas ruthenium red and cationized ferritin did. Isotope labelling after galactose oxidase treatment revealed that at least the carbohydrate portion of the major glycoproteins is present on the inner side of the granule membranes facing the content.     

NUCLEAR GLYCOCONJUGATES AND THEIR RELATION TO MALIGNANCY

1. The nucleus has a distinctive carbohydrate chemistry, the main features of which are the lack of glycosphingolipid, the high density of carbohydrate per unit area of nuclear membrane, the presence of glycosaminoglycan in the nuclear matrix and possibly the nuclear membranes, and the existence of glycosylated non-histone proteins. 2. The nucleus has considerable autonomy in its metabolism of glycosaminoglycan and has a capacity for glycosyl transfers involving glycosyl dolichyl phosphates and pyrophosphates. This latter activity probably resides in the nuclear membranes. 3. The soluble fraction of the nucleoplasm contains the total cellular CMP-sialic-acid synthetase and, hence, all sialic acid metabolism passes through the nucleus, which may have a regulatory role. Uncertainty remains as to the sialic acid content of the glycoproteins of the nucleus and it is likely to vary between cell types. 4. Malignancy is associated with several alterations in the glycosylation of nuclear membranes, including increased levels of sialic acids in the glycoproteins of the inner nuclear membrane: changes in glycosylation of the matrix and chromatin are not yet well defined. In malignancy, some nuclear glycoproteins may possibly appear in other cellular membranes.     

Origin of the minor glycoproteins of murine leukemia viruses.

Polyacrylamide gel electrophoretic analysis and immunoprecipitation were used to study glycoproteins from purified Rauscher murine leukemia virus (R-MuLV) and from AKR thymic lymphoblastoid cell membranes. In addition to gp70, a minor glycoprotein of approximately 52,000 daltons (gp52) was demonstrated in purified R-MuLV preparations, which was antigenically related to gp70. Analysis of R-MuLV glycopeptides obtained after exhaustive Pronase digestion showed that gp70 has at least two different glycopeptide size classes with molecular weights of 5,100 and 2,900, respectively. gp52, however, contained only a single glycopeptide size class of approximately 5,100 daltons, indicating that the two glycoproteins contain distinct carbohydrate components. Trypsin treatment of R-MuLV converted gp70 into a product with a molecular mass of approximately 52,000 daltons as well as a 45,000-dalton minor product, with little effect on virus infectivity. Similarly, trypsin treatment of 125I-labeled glycoproteins derived from AKR mouse lymphoblastoid cell membranes generated fragments antigenically related to gp70 and similar in size to those obtained by trypsin treatment of R-MuLV. In both cases, the appearance of cleavage products was accompanied by a decrease in gp70 during trypsin treatment. The occurrence of glycosylated components antigenically related to gp70 in AKR membrane glycoprotein preparations and in purified R-MuLV preparations which were similar to those generated by trypsin treatment supports the concept that these minor components arise from proteolytic cleavage of gp70.     

Galactose transfer in the membranes of human milk fat globules (author's transl)]

Galactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-acetylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when alpha-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency, This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N-acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.     

Structural and quantitative changes of carbohydrate chain of erythrocyte membrane glycoproteins in experimental diabetes mellitus after treatment with agmatine

It was shown that the development of experimental diabetes mellitus accompanied by increase of desialylation of carbohydrate determinants of erythrocytes membrane glycoproteins, removal of both O-linked and N-linked oligosaccharides from the glycoproteins and decrease of erythrocyte membrane negative charges. Treatment of streptozotocin-induced diabetic rats with agmatine led to enhance the content of N-and O-glycans in the erythrocyte glycoproteins, increase in sialic acid content and restore the negative charge of the cell membrane. Detected changes in configuration of membrane components of red blood cells in diabetic animals after treatment with agmatine indicate circulating in the bloodstream cells with a repertoire of adhesion molecules and glycoprotein receptors, which are inherent to the population of young erythrocyte.     

A study of intracellular transport of secretory glycoproteins in rat liver

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.     

The binding of d-glucosyl-neoglycoproteins to the hepatic asialoglycoprotein receptor

The binding of D-glucosyl-neoglycoproteins and D-galactose-terminated glycoproteins to the hepatic asialoglycoprotein receptor of rabbit liver membranes were characterized and compared. The binding of both types of glycoproteins showed the same dependence on calcium concentration, sensitivity to neuraminidase, and degree of inhibition by various carbohydrate derivatives. These results, along with the observation that the rabbit liver membranes bound both the D-glucosyl- and D-galactosyl-terminated glycoproteins to the same extent, indicated that both types of glycoproteins bound to the same receptor. To confirm this hypothesis, receptors were isolated from rabbit livers by affinity chromatography using D-galactosyl-bovine serum albumin or D-glucosyl-bovine serum albumin immobilized on Sepharose. These receptors were shown to be identical by several chemical and immunological criteria as well as in their ability to bind equal amounts of D-galactosyl- and D-glucosyl-terminated glycoproteins. The conclusion is that the rabbit hepatic asialoglycoprotein receptor cannot discriminate between D-galactosyl and D-glucosyl-terminated glycoproteins and binds both.     

Plasma membrane glycoproteins of tumour cells in enzootic bovine leukosis compared with normal bovine lymphoid cells

Plasma membranes from tumor cells in enzootic bovine leukosis and from normal bovine lymphoid cells of different sources have been isolated and characterized. The glycoprotein composition of the different membranes has been studied by SDS-PAGE and by analysis of the carbohydrate composition as well as by lectin binding of glycoprotein fractions obtained by phenol treatment of the membranes. No differences in the electrophoretical glycoprotein pattern could be detected comparing (nonmalignant) cells from persistent lymphocytosis and tumour cells, suggesting that malignancy is not associated with the gain or loss of a major glycoprotein. A 151.5 kD glycoprotein, present in membranes from normal lymph node lymphocytes, seems to be absent in the membranes of peripheral blood lymphocytes as well as of tumour cells. The loss of this glycoprotein might thus be associated with a loss of sessility of bovine lymphoid cells. The carbohydrate analysis and lectin binding of extracted glycoproteins revealed a decreased fucosylation accompanied by an increased exposure of galactose residues as well as a loss or a decreased complexity of N-glycosidically bound oligosaccharides in the tumour cell glycoproteins compared with those of normal cells. These findings are discussed with regard to disturbed growth regulation of leukosis tumour cells.     

Glycoproteins and glycolipids of oxyntic cell microsomes I. Glycoproteins: Carbohydrate composition,analytical and preparative fractionation

Isotopically-labeled sugars were incorporated into glycoproteins of isolated bullfrog gastric mucosa. The majority of the label was found in gastric microsomal fractions which were shown to contain membranes derived from the oxyntic cell tubular membrane system and were not significantly contaminated with mucus. The tubular membranes contained exceptionally large quantities of carbohydrate (approx. 260 μg/mg protein). Most of the sugar (73%) was associated with protein in the following molar ratios: hexose, 1.0: fucose, 0.42; hexosamine, 0.62; sialic acid, <0.02. The remaining sugar, predominantly hexose, could be extracted into lipid solvents and was presumably glycolipid.Gastric microsomes were dissolves in sodium dodecyl sulfate and subjected to acrylamide gel electrophoresis and Sephadex G-200 fractionation. The latter preparative procedure yielded several molecular weight classes, each of which contained different sets of proteins and/or glycoproteins; however, the molar ratios of the sugars found in the two carbohydrate containing classes were quite similar.Significant quantities of carbohydrate were also found in gastric microsomal fractions from other species, e.g. pig and rabbit. Furthermore, characteristic proteins and glycoproteins were not present in tadpole gastric microsomes until the later stages of metamorphosis when HCl secretory capability had been established. The above findings suggest that glycoproteins may play an important role in oxyntic cell functions; the possibility of a membrane protective role is discussed.     

Study of membrane glycoproteins of human erythrocytes using lectins

A method to study the glycoprotein composition of cell membranes, in particular of human red blood cells, has been developed. It includes the separation of membrane components by the SDS-polyacrylamide gradient slab gel electrophoresis, electroblotting of the phoretograms onto the nitrocellulose sheets and detection of glycoprotein fractions with FITC and peroxidase labeled lectins. PNA detected asialoglycoproteins with O-linked oligosaccharide chains, corresponding to all the PAS-positive bands of the phoretogram. SBA interacted more selectively and revealed only certain PAS-positive bands. Glycoproteins with N-linked carbohydrate chains were PAS-negative and can be identified only by the interaction with WGA, LCL, RCA. Group-specific agglutinins have shown that the ABO antigenic determinants are located in N-linked carbohydrate chains of membrane glycoproteins.     

Topological and regulatory aspects of dolichyl phosphate mediated glycosylation of proteins

From the time of their synthesis in the rough endoplasmic reticulum until they are secreted, packaged in lysosomes, or appear as membrane components at the cell surface, the polypeptide chains of N- and O-linked glycoproteins remain associated with intracellular membranes that are components of the secretory pathway. The various co-translational and post-translational modifications of the carbohydrate moieties of glycoproteins have been shown to occur within morphologically and functionally distinct regions of this complex membrane system. However, the sugar nucleotides, which serve as precursors to the oligosaccharide moieties of these glycoproteins, are synthesized almost exclusively in the cytoplasm. These findings raise a number of questions about the mechanisms involved in the transmembrane assembly of membrane and secretory glycoproteins. In this paper these questions are reviewed and recent studies directed towards providing answers to them are summarized. In addition, information related to the possible role of dolichyl phosphate in regulating the glycosylation of proteins is presented.     

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes.

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuosus polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrance and of the whole platelet components established in this system.     

Glycoproteins and glycopeptides in the chromaffin granule membrane

The glycoproteins and glycopeptides of adrenal chromaffin granule membranes have been analyzed by gel filtration, electrophoresis, and amino acid analysis. It has been found that almost all of the polypeptides present in the membrane are glycoproteins. Indeed, most of these possess sugar specificities which permit binding to concanavalin A-Sepharose. A new set of low-molecular-weight glycopeptides was found. There is an inverse correlation between carbohydrate content and polypeptide molecular weight.This research was supported by grant NS-13201 from the National Institutes of Health.     

Phosphatidylcholine-induced repair of damaged hepatocyte membranes in heliotrine poisoning

Hepatocyte membranes destruction in experimental toxic hepatitis caused by heliotrine administration was accompanied by a 10-fold increase in blood serum activity of aldolase fructose-I-monophosphate, a decrease in cytochrome P-450 content, an increase in the rate of cytochrome P-450 inactivation, as well as a decrease in microsomal glucose-6-phosphatase activity. Administration of phosphatidylcholine liposomes decreased the activity of aldolase twofold, which indirectly shows partial reconstitution of liver cell membranes. Phosphatidylcholine protective action is also manifested in an increase in the activity of glucose-6-phosphatase, a microsomal marker enzyme, up to its control level and in a 20% reduced rate of cytochrome P-450 inactivation. It has been shown that destroyed liver cell membranes may be repaired by the introduction of phosphatidylcholine in the form of multilayer liposomes.