Showing papers on "Sialic acid published in 1983"

Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity

Abstract: The haemagglutinin (HA) glycoproteins of influenza virus membranes are responsible for binding viruses to cells by interacting with membrane receptor molecules which contain sialic acid (for review see ref 1) This interaction is known to vary in detailed specificity for different influenza viruses (see, for example, refs 2–4) and we have attempted to identify the sialic acid binding site of the haemagglutinin by comparing the amino acid sequences of haemagglutinins with different binding specificities We present here evidence that haemagglutinins which differ in recognizing either NeuAcα2→3Gal- or NeuAcα2→6Gal-linkages in glycoproteins also differ at amino acid 226 of HA1 This residue is located in a pocket on the distal tip of the molecule, an area previously proposed from considerations of the three-dimensional structure of the haemagglutinin to be involved in receptor binding5

Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule

Abstract: The neural cell adhesion molecule, N-CAM, is a cell surface glycoprotein found on embryonic and adult neurons and on a variety of ectodermal and mesodermal tissues in very early embryos. During development, it shows local variations in prevalence at the cell surface as well as conversion from an embryonic form (E form) with high sialic acid content to an adult form (A form) with lesser amounts of this sugar. This E leads to A conversion occurs on different schedules in different brain regions, and it has been hypothesized that both the conversion and the prevalence changes are related to early regulation of pattern formation and connectivity. In order to identify precisely the consequences of these mechanisms of local cell surface modulation of N-CAM, an assay was developed to measure the rate of aggregation either of vesicles reconstituted from lipid and purified N-CAM or of native brain membrane vesicles. In both preparations, aggregation was greater than 95% inhibitable by specific anti-(N-CAM) Fab' fragments. The rates of aggregation of reconstituted N-CAM vesicles and native brain vesicles were found to be inversely related to the sialic acid content of their N-CAM molecules, with full desialylation resulting in about a 4-fold increase in rate over E-form N-CAM. Intermediate rates were obtained both with A-form N-CAM (which contains only one-third of the sialic acid content of E-form N-CAM) and with partially desialylated E-form N-CAM. The rate of coaggregation of reconstituted vesicles containing E-form N-CAM with reconstituted vesicles containing A-form N-CAM was also intermediate, implying that desialylation did not change the nature of (N-CAM)-(N-CAM) binding but only its rate. Even larger alterations in vesicle aggregation rate were seen when the amount of N-CAM per vesicle was altered. A 2-fold increase in the N-CAM-to-lipid ratio of reconstituted vesicles resulted in a greater than 30-fold increase in their rate of aggregation. Moreover, desialylation did not cause a further increase in the rate of aggregation of these already rapidly aggregating vesicles. These results in a model system demonstrate the large range of binding rates that are obtainable by various forms of local surface modulation of N-CAM. They are consistent with the proposal that similar alterations affecting (N-CAM)-mediated cell adhesion in vivo may be major factors in pattern formation during development of the nervous system.

Characterization of the human receptor for T-cell growth factor.

Abstract: Anti-Tac monoclonal antibody has been identified as a putative antibody against the receptor for T-cell growth factor (TCGF) We now show that: (i) TCGF blocks 85% of 3H-labeled anti-Tac binding to phytohemagglutinin-activated lymphoblasts and (ii) both anti-Tac and anti-TCGF immunoprecipitate a protein band that appears to represent TCGF crosslinked to its receptor on HUT-102B2 cells In HUT-102B2 cells, the TCGF receptor is a Mr 50,000 glycoprotein with internal disulfide bond(s) and a pI of 55-60, and it represents approximately equal to 005% of total cellular de novo protein synthesis It contains a peptide of Mr 33,000 that is processed to a mature form that includes N-linked and O-linked sugars and sialic acid

Lectin receptors as lymphocyte surface markers

Abstract: Publisher Summary The lectin receptors are characteristic markers of distinct lymphocyte subpopulations in mouse, man, and several other animals. Just like antigenic surface markers, lectin receptors are extremely useful for the identification and separation of lymphocytes, and therefore for studies on the functions and lineages of cells with a given phenotype. Some of them may even be considered as differentiation markers. Purification procedures designed to recover a representative culture of lymphocytes from blood or other tissues may lead to the removal of less well-defined subpopulations of these cells. In addition, cell fractionation may lead to alterations in cellular constituents, as shown by the finding of a marked increase in potassium and a decrease in sodium ion content in isolated rat thymus cells and compared to the levels of these ions in rapidly excised whole thymus. Two lymphocyte subpopulations are easily distinguished in the thymus, one located in the cortex and the other in the medulla. The medullary thymocytes, which in the past could be isolated in sufficient quantities only from mice that had been treated with cortisone or radiation, comprise approximately 10% of the total number of thymus cells. It has also been reported that cortisone resistant thymocytes and spleen T cells are more negatively charged and have a higher content of sialic acid than unfractionated thymocytes.

Shedding and immunoregulatory activity of YAC-1 lymphoma cell gangliosides.

Abstract: YAC-1 lymphoma cells, both when cultured in vitro and when passaged in ascites form in vivo , synthesize gangliosides (means of 22.1 and 14.7 nmol lipid-bound sialic acid isolated per 108 cells, respectively) with potent inhibitory effects on mitogen- and antigen-induced lymphoproliferation: 10 to 30 nmol highly purified YAC-1 gangliosides/ml caused >90% inhibition of proliferative responses of murine lymphocytes to concanavalin A, lysozyme (a soluble specific antigen), and allogeneic cells (mixed-lymphocyte response). Measurable quantities of these gangliosides were shed by the tumor cells in vitro and also were recovered from the ascites fluid in vivo . Furthermore, the gangliosides isolated from ascites fluid (mean of 15.3 nmol/ml) had inhibitory activity of a magnitude similar to that of the gangliosides isolated from the tumor cells. Therefore, significant inhibition of normal lymphoproliferative responses by tumor-derived gangliosides occurred at ganglioside concentrations which are actually present in the fluid surrounding the tumor cells in vivo . These results support the hypothesis that shedding of gangliosides may serve to protect tumor cells from host immune destruction.

Molecular topography of the neural cell adhesion molecule N-CAM: surface orientation and location of sialic acid-rich and binding regions

Abstract: Chemical analyses and binding studies have been correlated to clarify the relationship of structure to function in the neural cell adhesion molecule (N-CAM) from embryonic chicken brain. N-CAM isolated from the cell surface appears to include two closely related polypeptide chains. Treatment with neuraminidase of such preparations of N-CAM bound by antibodies on solid supports yielded components of Mr 140,000 and 170,000. These components each had the same amino-terminal sequence as N-CAM and gave nearly identical profiles on peptide maps. Immunoprecipitation of N-CAM from 9-day brain cells treated with tunicamycin yielded corresponding components of Mr 130,000 and 160,000, suggesting that the differences between these two components of N-CAM are in the polypeptide rather than the carbohydrate portions of the molecules. N-CAM appears to be oriented with the amino terminus extending away from the cell surface and with the bulk of the sialic acid near the middle of the peptide chain. As shown previously, incubation of N-CAM at 37 degrees C generates a fragment (Fr1) of Mr 65,000 that lacks most of the sialic acid. Treatment of membranes with Staphylococcus aureus V-8 protease released a fragment (Fr2) of N-CAM that contained most of the sialic acid; this fragment had an Mr of 108,000 after neuraminidase treatment. Both of these fragments contain the amino-terminal portion of the polypeptide chain. At least a portion of the N-CAM binding site was found to be located in the amino-terminal region of the peptide chain. Most or all of the sialic acid was not directly involved in binding, although it can influence binding, as indicated by the finding that neuraminidase-treated N-CAM (desialylated-N-CAM) bound to cells to a greater extent than untreated N-CAM. The Fr1 and the Fr2 fragments in solution did not bind to cells but were as effective as N-CAM and desialylated-N-CAM as competitors for N-CAM binding to cells. When fixed covalently to beads, N-CAM, desialylated-N-CAM, and the Fr1 and Fr2 fragments bound specifically to cells. In contrast, the N-CAM autolysis products released along with Fr1 neither bound to cells nor competed for N-CAM binding. In addition to suggesting a location for the N-CAM binding region, the accumulated results raise the possibility that valence may play a key role in N-CAM binding.

A developmentally regulated neuraminidase activity in Trypanosoma cruzi

Abstract: The human pathogen Trypanosoma cruzi (Y strain) contains a neuraminidase activity that varies widely in the different developmental stages of the parasite. The specific neuraminidase activity of infective trypomastigotes obtained from tissue culture and from the bloodstream of infected mice is 7 to 15 times higher than that of the acellular culture forms. Amastigotes were devoid of enzyme activity. The enzyme has a pH optimum of 6.0 to 6.5. Live trypanosomes released sialic acid from human erythrocytes and plasma glycoproteins. Several sialyl compounds were hydrolyzed by the parasite, but the best substrate was the protein orosomucoid. Erythrocytes from infected mice with T. cruzi parasitemia were agglutinated by peanut lectin and the hemagglutination titer was correlated with the degree of parasitemia.

Ganglioside‐Induced Neuritogenesis: Verification That Gangliosides Are the Active Agents, and Comparison of Molecular Species

Abstract: Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility that neurite-stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three-step purification procedure that eliminated at least 95% of the contaminating peptides. These purified preparations retained their capacity to induce extensive neurite growth in neuro-2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of their inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.

Evidence for mucins and sialic acid as receptors for Pseudomonas aeruginosa in the lower respiratory tract.

Abstract: The nature of the receptors for mucoid and nonmucoid Pseudomonas aeruginosa was investigated by using adherence to injured tracheal epithelium as a model. Bovine submaxillary mucin and crude rat tracheal mucin inhibited the adherence of both types of P. aeruginosa. Among the sugars present in these mucins only N-aceylneuraminic acid inhibited adherence. Inhibition of adherence probably involved the binding of N-acetylneuraminic acid to the bacterial cells and not to the tracheal cells. The mucoid strain appeared to be much more sensitive to inhibition by N-acetylneuraminic acid. Periodate oxidation and cholera filtrate also reduced the adherence of both strains, but Clostridium perfringens neuraminidase treatment did not alter adherence. A nonmucoid isogenic mutant of an unstable mucoid strain was also inhibited by N-acetylneuraminic acid. These data suggest that the receptor for P. aeruginosa is a sialic acid moiety on cell surfaces or in mucins.

Metastatic potential severely altered by changes in tumor cell adhesiveness and cell-surface sialylation.

Abstract: A plastic adherent variant line (ESb-M) of a highly invasive and metastatic murine T cell lymphoma (ESb) was found to have lost its metastatic potential while still being tumorigenic in normal syngeneic hosts. The variant retained most of its ESb-derived antigenic and biochemical characteristics but differed at binding sites for certain lectins with specificity for terminal N-acetylgalactosamine residues. Whereas such sites were masked by sialic acid on metastatic ESb cells, they became unmasked on the adherent variant line. Metastatic revertants of ESb-M cells did not express the respective lectin receptor sites because these were again masked by sialic acid. It is suggested that the masking of specific lectin receptors sites on the tumor cell surface is of crucial importance for metastatis. If freely exposed, these sites may change adherence characteristics of the cells possibly not only in vitro (to plastic) but also in vivo.

Inhibition of enterotoxin from Escherichia coli and Vibrio cholerae by gangliosides from human milk.

Abstract: Inhibitory activity of enterotoxin from Escherichia coli and Vibrio cholerae was associated with the ganglioside fraction of human milk. Both the milk fat and skim milk contained gangliosides that inhibited the toxins. The most purified milk fraction contained three glycolipid components, of which two migrated close to ganglioside GM1 on thin-layer chromatography plates. A component with a slightly different mobility from GM1 appeared to be associated with the inhibitory activity. Milk ganglioside fraction, derived from 2 ml of human milk, contained 1 to 4 micrograms of lipid-bound sialic acid and completely inhibited 0.1 micrograms of cholera toxin in rabbit intestinal loop experiments. It is suggested that human milk gangliosides, although present only in trace amounts, may be important in protecting infants against enterotoxin-induced diarrhea.

Analysis of maternal IgG subpopulations which are transported into the chicken oocyte.

Abstract: Chicken serum and oocyte IgG were compared. Purified intact IgG and mercuripapain-produced Fc fragments of yolk and serum IgG were analysed by isoelectric focusing. All IgG bands were identical, indicating that all subpopulations of serum IgG were present in the yolk. Upon papain hydrolysis of both serum and yolk IgG, four identical Fc bands were produced from all serum and yolk samples. Sialic acid measurements showed that there was no significant difference in sialic acid content between serum and oocyte IgG. From these results we conclude that: (i) ovarian IgG receptor(s) selectively transports all subpopulations of maternal IgG; (ii) there is no selective destruction of IgG during transport; and (iii) yolk IgG has the same amount of sialic acid as the serum IgG.

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Abstract: Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.

Role of the liver in clearance and excretion of circulating carcinoembryonic antigen (CEA).

Abstract: CEA is a glycoprotein with a molecular weight of 200,000 containing 55%–65% carbohydrate. The removal of only two sialic acid residues result in rapid uptake from the circulation by the liver and catabolism in the lysosomes. There is a receptor on the plasma membrane of the hepatocyte (hepatic binding protein) which recognizes galactosyl residues. About 70% of125I-labeled intact CEA is cleared by the liver in 1 hr. The exposure of terminal galactose residues by removing sialic acids determines the rate of clearance. CEA is probably initially taken up by Kupffer cells and transferred to hepatocytes. About 10% of CEA added to an isolated perfused liver appears in bile. Biliary duct obstruction and cholestasis may elevate plasma CEA levels due to detergent effects on the liver cell receptors.

Salla disease A new lysosomal storage disorder with disturbed sialic acid metabolism

Abstract: Salla disease is a lysosomal storage disorder associated with increased urinary excretion of free sialic acid. The main clinical features in 34 patients were severe psychomotor retardation of early onset, ataxia, athetosis, rigidity, spasticity, and impaired speech. Growth retardation, thick calvarium, and exotropia were present in about half the patients. The amplitude of EEG decreased progressively with increasing age. Life span appears to be normal; the age range of the patients was 3 to 63 years. Genealogic studies suggest an autosomal mode of inheritance. A thin-layer method is described for the detection of increased urinary free sialic acid excretion. The basic defect is so far unknown.