Showing papers on "Sialic acid published in 1975"

Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain.

Abstract: The distribution, carbohydrate composition, and metabolism of glycoproteins have been studied in mitochondria, microsomes, axons, and whole rat brain, as well as in various synaptosomal subfractions, including the soluble protein, mitochondria, and synaptic membranes. Approximately 90% of the brain glycoproteins occur in the particulate fraction, and they are present in particularly high amounts in synaptic and microsomal membranes, where the concentration of glycoprotein carbohydrate is 2-3% of the lipid-free dry weight. Treatment of purified synaptic membranes with 0.2% Triton X-100 extracted 70% of the glycoprotein carbohydrate but only 35% of the lipid-free protein residue, and the resulting synaptic membrane subfractions differed significantly in carbohydrate composition. The glycoproteins which are not extracted by Triton X-100 also have a more rapid turnover, as indicated by the 80-155% higher specific activity of hexosamine and sialic acid 1 day after labeling with [3H]glucosamine in vivo. The specific activity of sialic acid in the synaptosomal soluble glycoproteins 2 hr after labeling was greater than 100 times that of the synaptosomal particulate fraction, whereas the difference in hexosamine specific activity in these two fractions was only twofold, and by 22 hr there was little or no difference in the specific activities of sialic acid and hexosamine in synaptosomal soluble as compared to membrane glycoproteins. These data indicate that sialic acid may be added locally to synaptosomal soluble glycoproteins before there is significant labeling of nerve ending glycoproteins by axoplasmic transport. Fifty to sixty percent of the hyaluronic acid and heparan sulfate of brain is located in the various membranes comprising the microsomal fraction, whereas half of the chondroitin sulfate is soluble and only one-third is in microsomal membranes. When microsomes are subfractionated on a discontinuous density gradient over half of the hyaluronic acid and chondroitin sulfate are found in membranes with a density less than that of 0.5 M sucrose (representing a six- to sevenfold enrichment over their concentrations in the membranes applied to the gradient), whereas half of the heparan sulfate is present in membranes with a density greater than that of 0.8 M.

Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

Abstract: The carbohydrate moiety of carcinoembryonic antigen could be sequentially degraded by repeated cycles of periodate oxidation, reduction, and mild acid hydrolysis (Smith degradation). After three complete degradations, all fucose and sialic acid, 80% of the galactose, 65% of the mannose, and about 40% of the N-acetylglucosamine were eliminated without impairing the ability of degraded carcinoembryonic antigen to react with specific antisera against the antigen. Inhibition studies in a carcinoembryonic antigen/rabbit anti-carcinoembryonic antigen precipitating system with oligosaccharides covering previously known internal structures of glycoproteins and presumably corresponding to the internal carbohydrate region of the antigen, demonstrated that none of the compounds tested was inhibitory. Nor could any inhibitory effect on the binding of carcinoembryonic antigen to antibody against the antigen in a radioimmunoassay system be domonstrated for the carbohydrate moiety prepared by hydrazinolysis or the glyco peptide fraction isolated after papain degradation of the antigen. However, if carcinoembryonic antigen is completely reduced and alkylated, with three intrachain disulfide bonds cleaved per 10-5 g, the immunological activity is reduced to 3-5% of untreated antigen. Furthermore, treatment of the antigen with 0.5 NaOH at 20 degrees for 2 hr completely abolished its ability to react with antiserum, whereas its ability to precipitate with a series of lectins was unchanged. No release of low-molecular-weight carbohydrate orchange in sugar composition of alkali-treated antigen was observed. Our tentative conclusion is that the carbohydrate moiety of carcinoembryonic antigen does not contain the tumor-associated determinant(s).

Glycoprotein Metabolism in Inflammatory and Neoplastic Diseases of the Human Colon

Abstract: Summary Carbohydrate compositions of the membrane and cytoplasmic fractions of human normal and cancerous colonic mucosa were compared in patients with blood groups O and B The total sugar content in both fractions was reduced in the cancer tissues to about one-third of that in the normal colonic mucosa The sugars that are associated with mucinous glycoproteins such as fucose and N-acetylgalactosamine were reduced significantly, while sugars that are primarily associated with “serum-type” glycoproteins were relatively unchanged or reduced to a lesser extent The activities of glycoprotein:glycosyltransferases were variable, some showing no significant change, others being significantly reduced in cancerous tissues A polypeptidyl:N-acetylgalactosaminyltransferase (an enzyme that catalyzes the transfer of the first sugar to hydroxyamino acids of the protein core of mucinous glycoproteins), a sialytransferase (involved in the addition of sialic acid to mucinous glycoproteins), and a galactosyltransferase (thought to be responsible for blood group B antigenicity) were reduced in the cancerous colonic tissue In contrast, the activities of these glycosyltransferases were unchanged in the colonic mucosa of patients with granulomatosis or ulcerative colitis Glycosidase activities in the normal, cancerous, and inflammatory tissues were the same These results suggest that in colonic cancer tissues the synthesis of one type of oligosaccharide chain may be greatly affected, while another family of oligosaccharides may remain relatively unaffected

Molecular basis of Tn-polyagglutinability.

Abstract: Spectrophotometric and gas-liquid chromatographic analyses on the carbohydrate moiety of tryptic erythrocyte glycopeptides from persons with Tn-syndrome reveal a selective lowering of the galactose and sialic acid content, the degree being dependent on the percentage of polyagglutinable cells. Alkaline borohydride specifically releases N-acetylgalactosaminitol, and the amount is correlated to the percentage of pathological acetylgalactosaminitol, and the amount is correlated to the percentage of pathological erythrocytes. It is concluded that the alkali-labile carbohydrate chains of Tn-polyagglutinable red cells solely consist of N-acetylgalactosamine linked to serine or threonine. Experiments with heterophile agglutinins whose specificity is known are in line with the above-mentioned results. As judged from SDS-polyacrylamide gel electrophoresis the three major membrane glycoproteins are affected to a different extent by the defect.

Proteins specified by herpes simplex virus. XIII. Glycosylation of viral polypeptides.

Abstract: In the course of herpes simplex virus 1 (HSV-1) replication in human epidermoid carcinoma no. 2 cells, the synthesis and glycosylation of host cell proteins ceases and is replaced by the synthesis and glycosylation of virus-specified polypeptides. Analyses of the synthesis of viral glycoproteins show that the glycosylation of viral polypeptides occurs late in the virus growth cycle and that certain of the precursors to major vital glycoproteins are members of the gamma group of polypeptides, i.e., polypeptides synthesized at increasing rates until 12 to 15 h postinfection. Viral glycoproteins are formed by stepwise additions of heterosaccharide chains to completed precursor polypeptides. The precursor and the highly glycosylated product are separable by gel electrophoresis and are localized in different fractions of infected cells. Within 15 min of their synthesis, precursor polypeptides acquire heterosaccharide chains of about 2,000 molecular weight, which contain glucosamine but little or nor fucose or sialic acid. Both precursor and product of this first stage of glycosylation are absent or present in low concentrations in the surface membranes of the infected cell and in the virion. The partially glycosylated product is then conjugated further in a slow, discontinuous process to form the mature glycoprotein of the virion and plasma membrane. These mature products bear large heterosaccharide units with molecular weights greater than 4,000 to 5,000; these contain fucose and sialic acid as well as glucosamine. Heterosaccharide chains from infected and uninfected cells are distributed among discrete size classes and the smallest chains consist of multiple saccharide residues.

Sialic acid and the social beeiaviour of cells

Abstract: Summary 1. It is suggested that specific carbohydrate side-chains of membrane glycoproteins are the sites for cell recognition or adhesion when the terminal sugar, sialic acid, is absent. 2. It is suggested that sialic acid plays a ‘protective’ or ‘blocking’ role in cell interactions so that addition of sialic acid to asialo side-chains converts them to forms inactive for recognition. This principle of ‘blocking’ by sialic acid has been observed in other situations as in covering tumour antigens and in protecting glycoproteins from uptake by the liver. It is here extended to cell-cell adhesions. 3. It is to be expected that specific ‘protective’ actions of sialic acid in membrane-bound glycoproteins will be difficult to detect. As a charged residue, sialic acid is likely to have a strong influence both on the glycoproteins on which it is borne and on their interactions with each other at the cell surface. Removal of sialic acid by enzymes could therefore perturb the structure of the cell surface in several ways and so obscure the ‘protective’ effects of sialic acid. Sialic acid is therefore suggested to have a structural role also. 4. Evidence is assembled in favour of a model in which sialysation of specific adhesive receptors affects the social behaviour of cells. This may be an effect associated with growing cells since the contact properties of mitotic cells (and populations rich in dividing cells) are decreased by the increased sialysation of receptors. One of the factors associated with malignant behaviour could be that adhesive receptors are permanently blocked by sialic acid. 5. A schematic representation of some of the points is given in Fig. 4.

Isolation and characterization of two hydroxyproline-containing glycoproteins from normal animal lung lavage and lamellar bodies.

Abstract: Two glycopeptides, present in particulate material obtained by pulmonary lavage from normal rabbits, were isolated and characterized. The same two glycopeptides were present in preparations of lamellar bodies from rabbit lung. The estimated molecular weights of the two glycopeptides by sodium dodecyl sulfate-acrylamide gel electrophoresis were found to be 62,000 and 36,000 and both were found to contain hydroxyproline and relatively high amount of glycine (11 and 15 per cent, respectively). Carbohydrate analysis of the two glycopeptides demonstrated the presence of glucosamine, sialic acid, mannose, Fucose, and galactose. Similar glycopeptides of the same molecular weights, and amino acid and carbohydrate compositions have been found in layage mateial isolated from lungs of patients with alveolar proteinosis. The data indicate that thses two collagen-like glycopeptides are major intra-alveolar proteins in many mammals, including humans.

Antibodies reactive with cell surface carbohydrates.

Abstract: Normal and immune sera from various animal species were fractionated on columns of Sepharose covalently coupled with the glycoprotein fetuin. Elution of the material bound to fetuin yielded low but reproducible amounts of protein, ranging from 0.02 to 0.2% of the protein mass of the input sera. This material has been identified by immunoelectrophoresis in agar and by zone electrophoresis on cellulose acetate as immunoglobulin. The Ig fractions bound and agglutinated erythrocytes of various species, and also bound to cells from various mouse tissues including heart, kidney, thymus, and spleen. In all cases, the binding was inhibited by glycoproteins such as fetuin and thyroglobulin, by a glycopeptide isolated from fetuin, and by some bacterial lipopolysaccharides. When the binding of these Ig fractions to mouse splenocytes was tested in the presence of 17 saccharides, no inhibition of binding was observed except by sialic acid, D-galactose, N-acetyl-D-glucosamine, and D-mannose, all of which showed partial inhibition. Inasmuch as these four saccharides are present on the carbohydrate moiety of fetuin, the results suggest that the isolated material is a carbohydrate-specific Ig (CS-Ig) fraction of serum capable of binding to the carbohydrate portion of cell surface receptors and glycoproteins. When bound to lymphocytes, these CS-Ig molecules induced redistribution (patching and capping) of cell surface receptors. Moreover, the CS-Ig fractions from chicken and rabbit sera were weakly mitogenic for mouse splenic lymphocytes. CS-Ig fractions are useful new reagents for studying glycoproteins and the interactions and activities of cell surface carbohydrates.

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

Abstract: 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.

Sindbis virus glycoproteins: effect of the host cell on the oligosaccharides.

Abstract: Sindbis virus was grown in four different host cells and the carbohydrate portions of the glycoproteins were analyzed. Sindbis virus grown in BHK-21 cells has more sialic acid and galactose than Sindbis virus grown in chicken embryo cells. In other respects the carbohydrates from virus grown in these two hosts are very similar. Sindbis virus grown either in chick cells transformed by Rous sarcoma virus or in BHK cells transformed by polyoma virus was also examined. In comparisons of virus from normal and transformed cells, differences in the amount of sialic acid were observed; but otherwise the carbohydrate structures appeared basically similar. The growth conditions used for the host cell also affected the degree of completion of the carbohydrate chains of the viral glycoproteins.

Interactions between aerolysin, erythrocytes, and erythrocyte membranes.

Abstract: Aerolysin, a hemolytic and lethal exotoxin of Aeromonas hydrophila, was analyzed for amino acids Assuming 8 histidine residues/mol, the purified toxic protein has, by summation, a molecular weight of 49,000, a value in agreement with earlier estimates by other methods Erythrocytes from different animal species differ greatly in sensitivity to aerolysin's lytic action There is some correlation between sensitivity and phosphatidyl choline content Erythrocyte membranes of different species bind the toxin, and the efficiency of binding is a function of sensitivity to lysis Binding is temperature independent, is not dependent upon membrane sialic acid, and is decreased by prior treatment with phospholipase C and proteases Preparations of aerolysin convert substantial amounts of membrane phosphorus to water-soluble form; the conversion is concentration and temperature dependent Most of the conversion is attributable to contaminating phospholipase(s) that is separable from the toxin Aerolysin purified by electrophoresis in polyacrylamide gel retains some phospholipase activity, and this activity may or may not be a contaminant

Structures of gangliosides from bovine adrenal medulla.

Abstract: Five gangliosides, accounting for over 95% of the total ganglioside fraction, were isolated from bovine adrenal medulla by preparative thin-layer chromatography and the carbohydrate structures determined by a combination of periodate oxidation and permethylation techniques. Partially methylated alditol acetates were generated from the neutral sugars of the fully methylated glycolipids and identified by gas-liquid chromatography. Substitution on N-acetylgalactosamine was determined by methanolysis of the permethylated ganglioside, acetylation of the products, and identification of the resulting substituted methyl glycosides by GLC. Periodate oxidation followed by borohydride reduction confirmed some of the linkages and demonstrated the absence of (2-8) linkages between sialic acid units. Mass spectrometry of the permethylated gangliosides gave information on sugar sequence at the nonreducing end.

Evidence that the major cell suface glycoprotein of the TA3-Ha carcinoma contains the Vicia graminea receptor sites.

Abstract: The Vicia graminea lectin receptor of the nonstrain-specific TA3-Ha mammary carcinoma ascites cell of the strain A mouse was shown to be predominantly or exclusively on a large mucin-type surface glycoprotein. TA3-Ha cells adsorbed the lectin in amounts equivalent to 5-9 mg of this glycoprotein/10-9 cells, which was 100-400 times greater than by the strain-specific TA3-St cell, employed as a control. Release of sialic acid by incubation with neuraminidase increased the adsorptivity of the TA3-Ha cell three to fourfold and of the TA3-St cell six- to ten fold. Proteolysis of TA3-Ha cells released into the supernatant solutions approximately the same amount of inhibitory activity, equivalent to approximately 5 mg of the glycoprotein and only 10 per cent of the original adsorptivity remained on the cells. By contrast, TA3-St cells released no detectable inhibitory activity into the medium when subjected to similar proteolysis, even after neuraminidase treatment. Upon fractionation of released material on gel columns, high-molecular weight material and activity were found in the same fractions, but purified samples differed significantly in specific activity and carbohydrate composition. Heterogeneity in the carbohydrate moieties of the macromolecules was further demonstrated by incubation of these samples with neuraminidase, which enhanced their inhibitory activities from two- to tenfold.

Cellular adsorption function of the sialoglycoprotein of vesicular stomatitis virus and its neuraminic acid.

Abstract: Exposure of vesicular stomatitis (VS) virions to neuraminidase resulted in loss of their ability to agglutinate goose erythrocytes and to attach to L cells concomitant with hydrolysis of sialic acid. These viral adsorptive functions were also destroyed by tryspsinization. Sialyl transferase resialylation in vitro of neuraminidase-treated VS virions restored their hamagglutinating and adsorptive functions almost to original levels. Erythrocyte and L cell receptors for attachment of VS virions were blocked by fully sialylated fetuin and by VS viral sialoglycopeptides. Smaller VS viral glycopeptides generated by extensive trypsinization were less effective inhibitors of hemagglutination than were larger glycopeptides; neuraminic acid and neuraminosyl lactose had no capacity to inhibit hamagglutination or adsorption of virus to L cells. These data suggest that cellular receptors for viral adsorption recognize sialoglycopeptides of a certain size. Neuraminidase desialylation did not significantly alter the isoelectric point of VS virions. Cells exposed to DEAE-dextran, trypsin, or neuraminidase showed significantly increased capacity to attach fully sialylated but not desialylated VS virions. Neuraminidase desialylation of L cells, Chinese hamster ovary cells, and Madin-Darby bovine kidney cells resulted in enhanced susceptibility to plaque formation by VS virus.

Isolation and identification of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid from the urine of a patient with sialuria

Abstract: -Acetylneuraminic acid preparations from the urine of a patient with sialuria contain 12% of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. This new human sialic acid was isolated by ion-exchange and partition chromatography. The structure has been elucidated by mass spectrometry and confirmed by comparison with the synthetic compound. The properties of this unsaturated sialic acid in the orcinol/Fe3+ /HCl and the periodic acid/thiobarbituric acid tests as well as in thin-layer and gas-liquid chromatography are described. It does not react with acylneuraminate pyruvate-lyase. The origin of this new human sialic acid is discussed.