Showing papers on "Sialic acid published in 1978"

Regulation by membrane sialic acid of β1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway

Abstract: Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO4). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and β1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by β1H. A 1% decrease in intact sialic acid was associated with a 1% decrease in β1H activity in decay-dissociation of membrane bound C3b,Bb. Because removal of the C8 and C9 carbon atoms from the polyhydroxylated side chain of sialic acid by oxidation with NaIO4 was functionally equivalent to removal of the entire sialic acid moiety, secondary effects of the latter reaction, such as diminution of the negative charge of the membrane or exposure of penultimate galactose residues, were not considered to be responsible for the altered activity of β1H. These studies suggest that facilitation, by membrane sialic acid residues, of the interaction between bound C3b and β1H is essential to prevent the particle from effectively activating the alternative pathway.

Ganglioside structures and distribution: Are they localized at the nerve ending?

Abstract: Gengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid-bound sialic acid exceeds that of the protein-bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.

Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans.

Abstract: Four highly purified salivary glycoproteins were used to study salivary-bacterial interactions. One pair of glycoproteins was mucin-like in composition, whereas the second pair was not. By an agglutination assay, it was found that only the mucin-glycoproteins agglutinated Streptococcus sanguis and S. mutans. Removal of sialic acid from these molecules resulted in a loss of agglutination of S. sanguis but not of S. mutans. The agglutination phenomenon was shown to require a salivary macromolecule of at least 150,000 daltons.

Oligosaccharide chains are trimmed during synthesis of the envelope glycoprotein of vesicular stomatitis virus

Abstract: The biosynthesis and maturation of the oligosaccharide moieties of the envelope glycoprotein of vesicular stomatitis virus were investigated in virus-infected HeLa and BHK21 cells after pulse labeling with [2-3H]mannose. Two major forms of the virus glycoprotein were detected by polyacrylamide gel electrophoresis, which appear to correspond to the viral glycoprotein with either "precursor" or "mature" oligosaccharide chains. The precursor chains in both HeLa and BHK21 cells infected with vesicular stomatitis virus obtained after a 30-min pulse were large oligomannose structures containing approximately 7--9 mannose residues as estimated by gel filtration analysis. The size of the oligomannose structures initially transferred to the protein may have been even larger. Mature, virus-size oligosaccharide chains, which could be detected after a 20- to 30-min delay, contained only three mannose residues and, in addition, contained branch structures terminating in sialic acid. A precursor--product relationship of these two forms of oligosaccharide chains was demonstrated by pulse--chase labeling of virus-infected HeLa cells. These studies indicated that the large oligomannosyl core structures initially added to the glycoprotein were being "trimmed" by the removal of mannose residues prior to (and/or during) the addition of the branch chains terminating in sialic acid.

Properties of isolated human alpha1-antitrypsins of Pi types M, S and Z.

Abstract: 1. alpha1-Antitrypsin contains a single thiol group partly blocked in native plasma and reactive after mild reduction. 2. Human alpha1-antitrypsins of Pi types F, M, S and Z have been isolated with native microheterogeneity using thiol-disulfide (SH-SS) interchange reactions utilizing the reactive thiol group. 3. The pI of the various microheterogeneous fractions are given for protein M. Stepwise desialylation of alpha1-antitrypsin indicates that the charge difference between the major fractions is one sialic acid residue between each. This is further supported by the pI changes obtained on substitution of the single thiol with positively or negatively charged compounds. 4. Desialyation of purified proteins from each Pi type converts the individual microheterogeneous fractions to one major fraction. The pI shift for the variants studied indicate a difference of plus or minus one or two charge units between protein M and the variants. 5. A difference of one sialic acid residue was obtained for proteins M and Z by the thiobarbituric assay, but stepwise removal of sialic acid with neuraminidase revealed almost identical stepwise change of pattern of both proteins indicating the same number of sialic acid residues. 6. Electrofocusing has been used to identify CNBr fragments from proteins M, S and Z. 7. An amino acid substitution has been found to be located in one of the eight CNBr fragments, glutamic acid in protein M is substituted by lysine in protein Z. 8. The average concentration of alpha1-antityprsin in plasma from healthy males was found to be 1.32 g/1.

Blood-Group ABH-Specific Macroglycolipids of Human Erythrocytes: Isolation in High Yield from a Crude Membrane Glycoprotein Fraction

Abstract: Highly glycosylated, water-soluble ABH-specific sphingolipids, designated macroglycolipids, were isolated in high yield, up to 5 mg per unit of blood, from the crude human-erythrocyte-membrane glycoprotein fraction which is obtained by extraction of the membranes with chloroform/methanol/water. Both serological tests and radioactive labelling experiments indicated that these substances, rather than the glycoproteins, are the principal ABH-components in this fraction. The activities of A-specific, B-specific and H-specific macroglycolipids were very high, approximately 0.1 microgram inhibiting four hemagglutinating doses of the respective agglutinating reagents, and were thus comparable to those of secreted blood-group ABH-specific glycoproteins. The substances were stable to mild alkaline conditions. They contained fucose, galactose, glucosamine, glucose, sialic acid, sphingosine and fatty acids; blood-group-A-specific substances contained, in addition, galactosamine. No amino acids were detected. Assuming one glycosyl residue per molecule, the average number of sugars in A and B macroglycolipids was 31, and their molecular weights approximately 6100. The presence of beta-D-galactosidase-labile and sialic acid residues indicated that these substances contain nonreducing termini additional to the ABH immunodeterminants. In the B macroglycolipid, the ratio between nonreducing terminal alpha-D-galactopyranosyl and beta-D-galactopyranosyl residues was 1.7:1.0. The macroglycolipids formed clear aqueous solutions at concentrations as high as 30 mg/ml, were insoluble in 60--70% aqueous ethanol, and did not migrate on thin-layer chromatography unless they were acetylated. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed the macroglycolipids to be a heterogeneous mixture migrating throughout most of the region in which the periodic acid/Schiff-positive membrane glycoproteins are found. On the basis of the evidence presented, it is concluded that macroglycolipids are the predominant ABH-specific component in human erythrocyte membranes, and that they most likely account for previous observations of ABH activity in membrane glycoprotein fractions.

Characterization of the Hanganutziu-Deicher (serum-sickness) antigen as gangliosides containing n-glycolylneuraminic acid.

Abstract: Gangliosides that possess the same sugar sequence but differing in the type of sialic acid (N-acetyl- or N-glycolylneuraminic acid) were compared for their reactivity with Hanganutziu-Deicher (‘serum sickness’) antibodies by double-diffusion gel precipitation tests. Only N-glycolylneuraminic acid containing gangliosides formed precipitation lines with Hanganutziu-Deicher antibodies, thus suggesting that Hanganutziu-Deicher antigens are gangliosides that contain N-glycolylneuraminic acid.

Characterization of the glucose transporter from human erythrocytes

Abstract: The D-glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394--7390) Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid-protein ratio of 14:1 by weight The KD (014 micrometer) and extent (11 nmoles/mg protein) for binding of 3H-cytochalasin B was determined by equilibrium dialysis Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower-molecular-weight protein This preparation contains only 24% acidic and basic amino acids The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid

Altered cell surface organization of gangliosides and sialylglycoproteins of mouse metastatic melanoma variant lines selected in vivo for enhanced lung implantation.

Abstract: Qualitative and quantitative 'analyses of cell surface Sialylglycoproteinsand glycosphingolipidsof in vivo se lected mouse melanoma variant lines that show either a high (F10) or low (F1) degree of lung implantation have been made in culture and in in wVo-growntumors. The following observations have been made concerning the cell surface characteristics of the two cell lines: (a) al though the total, protein-bound, and lipid-bound sialic acid is significantly decreased (20 to 35%), the cell sur face-exposedSialylglycoproteinsand gangliosidesshowed a moderate increase in F10 cells as compared to F1 cells; (b) surface glycoproteinsof melanoma cells were studied with different labeling techniques followed by polyacrylamide gel electrophoresis and fluorography.The majority of surface glycoproteins were shown to be sialylfucosylgalactosyl and/or galactosaminyl glycoproteins; (c) the high-lung-implantingF10 cells reveal certain Sialylglyco proteinswith molecular weights of 66,000 (in culture) and 97,000, 84,000, 74,000, and 66,000 (in vivo), as detected by the galactose oxidase method, that are absent or weakly labeled in F1 cells; (d) metabolic labeling with glucosamine followed by neuraminidase hydrolysis reveals that F10 cells contain 80% more neuraminidase-accessible total cell surface sialic acid, the majority of which was contributedby AcNeu-a-(2—>3)-Gal-/3-(1-»4)-GlcCer as com pared to F1 cells; (e) F10 cells showed an enrichment of the quantity of ceramide dihexoside and a 5- to 6-fold higher cell surface exposure compared to F1 cells; (b) dramatic differences in ganglioside profile were seen be tween the in vivo and in vitro growth of F1 and F10 cells. The relationship of altered cell surface architecture of sialic acid-containing components to lung-specific im

Abnormal sialic acid content of the dysfibrinogenemia associated with liver disease.

Abstract: To evaluate the possibility that the carbohydrate composition of fibrinogen may be altered in the dysfibrinogenemia associated with liver disease, we studied the sialic acid content of purified fibrinogen from 12 patients with liver disease and its relationship to the prolongation of the thrombin time. Purified fibrinogen showed that Aalpha-, Bbeta-, and gamma-chains when reduced and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited prolongation of the thrombin time similar to that of the plasma from which it was derived. Sialic acid content of the purified fibrinogen ranged from 12.7 to 71.4% higher in patient fibrinogens when compared to normal controls. A progressive delay in thrombin time was associated with increasing sialic acid content of the patient fibrinogen. Enzymatic removal of sialic acid from four of the abnormal fibrinogens resulted in a shortening of their thrombin times to the range of the desialylated normal control. Periodic acid-Schiff reagent stained only the Bbeta- and gamma-chains of the reduced patient fibrinogens after sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the excess sialic acid is located on these two chains. These studies demonstrate a biochemical alteration of the functionally abnormal fibrinogen found in some patients with liver disease, and indicate that the excess sialic acid plays an important role in the functional defect of this protein.

360-MHz 1H nuclear-magnetic-resonance spectroscopy of sialyl-oligosaccharides from patients with sialidosis (mucolipidosis I and II).

Abstract: 360-MHz proton nuclear magnetic resonance spectra were recorded of 10 sialyl-oligosaccharides isolated form urine of sialidosis patients. Their structures are related to the complex aspareagine-linked glydan chains of glycoproteins. By correlation of these spectra and comparison with spectra of reference glycopeptides and sialy-lactose isomers it was possible to assign all signals belinging to anomeric, mannose H-2, sialic acid H-3 and N-acetyl protons. The number of the constituting monosaccharide residues of the oligomers can be obtained by integration of the above-mentioned singnals. The chemical shifts of the anometic and mannose H-2 proteins give information about the type of glydan structure (mono-, bi-, triantennary) and the presence of terminal sialic acid at eachof the antennas. The chemical shfts of sialic acid H-3 protons are typical for sialic acid residues in 23 or 26 linkage ot galactose.

The structure and function of the myocardial cell surface.

Abstract: External to the lipid bilayer or unit membrane at the surface of the myocardial cell is the glycocalyx. This coat is approximately 50 nm thick and is composed for two layers, the surface coat (SC) and the external lamina (EL). The SC is an integral part of the sarcolemma and many of its glycoproteins penetrate into or through the lipid bilayer. The glycocalyx invaginates with the unit membrane to fill the transverse tubules of the cell. Both layers contain abundant fixed negatively charged sites and a prominent constitutent of both is sialic acid. Removal of sialic acid produces a large specific increase in sarcolemmal calcium permeability without perturbation of potassium permeability. Sialic acid also accounts for a component of negatively charged sites which, with other acidic mucopolysaccharides, contributes to cationic binding at the surface of the cell. Calcium bound at the surface seems to be of importance in the excitation-contraction (EC) coupling sequence whether as a source of "trigger" calcium for the sarcotubular system or as a direct activator of the myofilaments. The bound Ca appears to be in rapid equilibrium with Ca in the vascular and interstitial spaces and is the probable immediate source of the Ca that crosses the sarcolemma. The integrity of the glycocalyx appears to be necessary in the prevention of uncontrolled entry of Ca into the cell.

The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus.

Abstract: 1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15 X 10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6 X 10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5 X 10(6), which is halved to 0.76 X 10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.

Demonstration of T-Transferase Deficiency in Tn-Polyagglutinable Blood Samples

Abstract: The serum and red cell membranes from seven Tn individuals have been tentatively characterized for the UDPgalactose: N-acetyl-d-galactosamine-β-d-galactosyltransferase and UDPgalactose: N-acetyl-d-glucosamine-β-4-d-galactosyltransferase activities using p-Nitrophenyl-2-acetamido-2-deoxy-α-d-galactopyranoside and p-Nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside respectively as low molecular weight acceptors. In five cases, Tn-positive and Tn-negative red cells were at first separated by Polybrene differential aggregation. The following conclusions have been drawn. 1 The β-3-d and β-4-d-galactosyltransferases activities are found in serum and red cell membranes from all normal individuals. 2 Polybrene-positive cells (normal sialic acid content) from Tn bloods have either normal or higher β-3-d and β-4-d-galactosyltransferases activities. 3 Polybrene-negative cells (low sialic acid content) from Tn bloods have a selective deficiency in β-3-d-galactosyltransferase (T-transferase) activity, but normal or even increased β-4-d-galactosyl-transferase activity. 4 The serum from all Tn individuals behaves like normal sera in respect of the two galactosyl-transferase activities. The serum may also be used as source of enzyme for conversion in vitro of Tn to T-reactive erythrocytes. The above data strongly support the view that Tn-polyagglutination results from a somatic mutation in stem cells of haematopoietic tissue which involves a single genetic step in red cell glycoprotein synthesis. It also provides further evidence of a dual population of erythrocytes in each Tn blood sample and suggests that T-transferase found in serum is produced in unidentified cells of the organism. No difference has been noticed between apparently healthy Tn donors and Tn patients.

Difference in Form of Sialic Acid in Red Blood Cell Glycolipids of Different Breeds of Dogs

Abstract: Hematoside from dog erythrocyte membrane was previously considered to contain a mixture of N-acetyl- and N-glycolyl- neuraminic acids. However, the hematoside preparation used in the previous study was obtained from pooled blood of several dogs, and individual variation in hematoside was not examined. In this work, hematosides of erythrocytes from 31 mongrel dogs and 108 dogs of 23 breeds were examined individually by thin-layer chromatography, and the component sialic acids were analysed by gas-liquid chromatography. Individual dogs had either NAN-hematoside or NGN-hematoside: dogs with N-glycolyl-neuraminic acid also had a trace of N-acetyl-neuraminic acid, but dogs with N-acetyl-neuraminic acid had no detectable N-glycolyl-neuraminic acid. A few mongrel dogs, some Kai dogs, Kishu dogs, Japanese spaniels and most Shiba dogs had NGN-hematoside, whereas all European dogs had NAN-hematoside and no NGN-hematoside. From pedigrees of some families, inheritance of NGN-hematoside was found to be autosomal dominant. NGN-hematoside is possibly one of dog blood group substances. The sialic acid of delipidized ghost protein of dogs with NGN-hematoside was N-glycolyl-neuraminic acid, and that of dogs with NAN-hematoside was N-acetyl-neuraminic acid. The sialic acid of plasma protein was mainly N-acetyl-neuraminic acid in all dogs.

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes.

Abstract: Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.

The type-specific polysaccharides of Streptococcus suis.

Abstract: Streptococcus suis types 1 and 2 were subjected to digestion with lysozyme. Serologically type-specific capsular polysaccharides were isolated from the lysates by ethanol precipitation followed by Sepharose 6B chromatography. The purified type 1 polysaccharide has a Kd value of 0.074 on a Sepharose 4B column and contains galactose, glucose, N-acetyl glucosamine, N-acetyl galactosamine, and sialic acid in a molar ratio of 2.42:1.00:1.00:1.13:1.39. The type 2 polysaccharide has a Kd value of 0.185 and is composed of rhamnose, galactose, glucose, N-acetyl glucosamine, and sialic acid in a molar ratio of 1.07:3.17:1.00:0.94:1.00. A comparison is drawn between the type polysaccharides of S. suis and those of group B streptococci.

Characterization of a Novel Sugar Sequence from Rat-Brain Glycoproteins Containing Fucose and Sialic Acid

Abstract: The structure of a major fucose-containing sugar sequence of rat brain glycoproteins was investigated. Glycopeptides obtained by proteolytic digestion of the lipid-free residue of whole rat brain were fractionated by affinity chromatography on Sepharose bound to concanavalin A and wheat-germ agglutinin. An N-glycosidic glycopeptide fraction corresponding to about 65 % o’f the protein-bound fucose was obtained. Methylation analysis before and after treatment with a-fucosidase or mild acid demonstrated that a major proportion of fucose is linked to the C-3 of N-acetylglucosamine. Studies involving neuraminidase digestion, partial acid hydrolysis, chromium trioxide oxidation and uronic acid degradation revealed that the fucose-containing sugar sequence has the structure : AcNeu(a2- 3)Gal(b1- 4)GlcNAc(jZ 1)aFuc) The structure of the Gal@ 1-4) [Fuc(a1-3)]GlcNAc sequence was also confirmed by gas-liquid chromatography and mass spectrometry after preparation of an oligosaccharide derivative by specific degradation with nitrous acid deamination. The sugar sequence, which has not previously been described in glycoproteins, is a sialosylated derivative of the so-called X antigen structure and it accounts for most of the fucose present in brain glycoproteins. The sugar sequence occurs mainly in membrane-bound glycoproteins, but is also found in the soluble fraction.

Methods for Isolation and Analysis of Gangliosides

Abstract: Gangliosides were first isolated from the brain of a Niemann-Pick patient (Klenk, 1935) and later in much larger amounts from the brain of a Tay-Sachs patient (Klenk, 1939). Klenk (1942) then obtained the same substances from normal brain and found them to be concentrated primarily in gray matter. More recent investigations of mammalian brain (Suzuki, 1965a; Svennerholm, 1970a,b; Yu and Ledeen, 1970) have revealed gray matter levels of approximately 700–1000 μg lipid-bound sialic acid per gram of fresh tissue for a variety of species, with white matter concentrations one-third to one-fourth this amount. This differential led at first to the erroneous conclusion that gangliosides are unique to the neuron, but subsequent work demonstrated their presence in virtually all cellular and subcellular fractions of brain that have been carefully analyzed. They are widely distributed in peripheral nerve and extraneural tissues as well, generally at concentrations well below those of brain.