Substrate specificity of viral, bacterial and mammalian sialidases with regard to different N,O-acetylated sialic acids and GM1.
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Acetylation↗
Biomedical subjects
R Schauer
Publications and source records attributed to R Schauer.
[Significance of human salivary and mucosa mucins in the area of the upper respiratory and digestive tract].
The importance of the mucins in saliva and mucus for the mucous membranes of the aero-digestive tract is underlined. The biochemistry of the mucins is briefly talked over, with the biological significance of the neuraminic acid being especially evoked. Decisive functions of the mucins for the mucous membranes of the upper respiratory and alimentary tracts, such as the protective, lubrication and transport functions, as well as their contribution to the formation of surface tension are discussed. Some of the few known pathobiochemical relationships are described. Pathobiochemistry of the mucins may also contribute to a better understanding of unsettled oncological problems of the aero-digestive tract. Mucin changes due to noxae--as e.g. by alcohol--may lead to a reduction of the mucous and salivary protective functions. After restricted mucin protective function, noxae--such as tobacco smoke--may then exercise their cancerogenic effects on the then unprotected epithelium.
Digestive System Physiological Phenomena↗
New chromatographic system for the rapid analysis and preparation of colostrum sialyloligosaccharides.
A new thin-layer chromatographic system on silica gel for the separation of sialyloligosaccharides is described. Calibration of the system with standard milk and colostrum sialyloligosaccharides is presented. The use of the system in monitoring different oligosaccharides is demonstrated for the purification of bovine colostrum sialyllactose isomers and a commercial sialyllactose product, and is discussed with respect to other biological fluids. The large-scale preparation of pure sialyllactose isomers from bovine colostrum is achieved using an improved ion-exchange separation on Dowex 1-X2 (less than 400 mesh) employing isomolar elution at 20 mM for monosialyloligosaccharides and 200 mM for disialyllactose. The purification of four major monosialyltrisaccharides, the 2-3 and 2-6 isomers of N-acetylneuraminyllactose, N-glycolylneuraminyl2-3lactose and N-acetylneuraminyl2-6-N-acetyllactosamine, and the disialyltetrasaccharide di-N-acetylneuraminyllactose is reported. The detection and partial purification of three new minor monosialyloligosaccharides is described.
Animals↗
The release of N-acetyl- and N-glycolloyl-neuraminic acid from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidases.
A series of substrates, sialyl(2 leads to 6)GalNAc and ganglioside GM3, containing either N-acetylneuraminic acid (AcNeu) or N-glycolloylneuraminic acid (GcNeu), has been prepared. The trisaccharide GcNeu(2 leads to 3)lactose was preapred by ozonolysis of GcNeu-GM3, and the disaccharides AcNeu(2 leads to 6)GalNAc and GcNeu(2 leads to 6)GalNAc were isolated from bovine submandibular-gland mucin by alkali elimination. Sialidases from Newcastle-disease virus, fowl-plague virus, influenza virus A2, Clostridium perfringens, Vibrio cholerae, Arthrobacter ureafaciens and human liver lysosomes were studied with the above substrates and all showed poorer cleavage of GcNeu-containing substrates when compared with the corresponding AcNeu-containing compounds. This was reflected in the Km and Vmax. values of these sialidases. Differences between viral and bacterial sialidases could be detected on the basis of their kinetic constants and time curves of sialic acid release. Preferred release of AcNeu relative to GcNeu was also observed with bovine submandibular gland mucin and a mixture of human and porcine erythrocytes, macromolecular substrates containing both AcNeu and GcNeu. The significance of differential cleavage of AcNeu and GcNeu by sialidases is considered together with examples of the role of GcNeu in physiologicaL systems.
Animals↗
The efficacy of neuraminidase treatment in studies on red cell aging.
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Animals↗
Carbohydrate surface constituents of T cells mediating delayed-type hypersensitivity that control entry into sites of antigen deposition.
Peritoneal exudate T lymphocytes (PETLs) that mediate delayed-type hypersensitivity (DTH) to sheep red blood cells in mice were modified by in vitro treatment methods which modify surface carbohydrate constituents. Neuraminidase treatment resulted in the release of both N-acetylneuraminic acid and N-glycolyl-neuraminic acid, and periodate treatment in the formation of the corresponding C7 analogues. Treatment of PETLs with neuraminidase led to a transient reduction of DTH reactions in syngeneic cell recipients. After treatment with neuraminidase plus galactose oxidase, adoptive mediation of DTH was more markedly reduced. Incubation of PETLs with periodate caused a permanent loss of DTH transferring capacity. The oxidation-induced effects following treatment of PETLs with neuraminidase plus galactose oxidase or with periodate could be reversed by subsequent reduction with borohydride of the previously formed aldehyde moieties. Decreased DTH reactions observed after the various treatment procedures were paralleled by reduced immigration of PETLs into sites of antigen deposition, indicating that the reduction/oxidation state of cell surface carbohydrates is crucial for induction of inflammatory processes by T cells. Trapping of PETLs in the liver could not be the sole mechanism since neuraminidase- and periodate-treated PETLs, but not neuraminidase plus galactose oxidase-treated PETLs, accumulated in the liver. It therefore appears that alterations in the reduction/oxidation state of the cell surface can lead to unresponsiveness of T cells to inflammatory signals.
Animals↗
Studies on malaria in Papua New Guinea: comparison of the surface glycoproteins on red blood cells from infected and uninfected individuals.
The levels of erythrocyte membrane sialic acid from 17 patients with Plasmodium falciparum malaria and 1 with Plasmodium vivax malaria, in Papua New Guinea, have been compared with 9 uninfected controls. The amounts of radioactivity incorporated into the major erythrocyte glycoproteins by the periodate/NaB3H4 or galactose oxidase plus neuraminidase/NaB3H4 methods were unchanged by malaria infection. The electrophoretic mobilities of these proteins also were unaffected. Several new glycoprotein bands with molecular weights (mol. wt) of 160,000, 89,000, 46,000, 42,000 and 33,000 Daltons were labelled on the surface of erythrocytes from infected individuals; however, none of these bands appeared in all malarious samples. Sialic acid levels on the erythrocyte membrane were also measured by exhaustive neuraminidase treatment and quantitative assay of released sialic acid. The amount of sialic acid was raised in 1 infected individual, within the normal range for Europeans in 4 others, and below this range with 3 patients. Apparently, extensive removal or modification of sialic acid on the surface of uninfected erythrocytes does not occur in human malaria, in contrast to the results obtained in earlier studies with the lethal murine malarias.
Adolescent↗
Specificities of limulin and wheat-germ agglutinin towards some derivatives of GM3 gangliosides.
Lipid vesicles containing derivatives of GM3 ganglioside (II3-N-acetylneuraminosyllactosyl ceramide) were used to study the specificities of two lectins (limulin and wheat germ agglutinin) towards N-acetyl neuraminic acid and N-glycoloylneuraminic acid and some of their natural and chemically modified derivatives. The extent of the lectin binding to the gangliosides was related to the aggregation process of the lipid vesicles which was monitored as an absorbance increase. Limulin binds specifically to lipid vesicles containing N-glycoloyl derivatives of GM3. The hydroxyl group at C-4 and the carboxyl group of neuraminic acid have to be free for the binding to limulin. The side chain of neuraminic acid is not involved in the binding site of limulin. Wheat germ agglutinin binds to GM3 ganglioside only when the hydrophilic tail of the neuraminic group is cut off (C7 analogues). The acetamido group but not the carboxyl group is involved in the binding to wheat germ agglutinin. The wheat-germ-agglutinin-induced aggregation of vesicles containing derivatives of GM3-ganglioside is dependent on the pH, on the ionic strength and on the presence of Ca2+ ions. The dependence on ionic strength and Ca2+ is a consequence of the electrostatic repulsion of the vesicles. The wheat-germ-agglutinin-induced aggregation process of vesicles containing any suitable GM3-ganglioside derivative was reversed by the addition of N-acetylglucosamine showing that the N-acetylneuraminic acid derivatives bind to the N-acetylglucosamine binding site.
Calcium↗
Uptake, metabolism and excretion of orally and intravenously administered, 14C- and 3H-labeled N-acetylneuraminic acid mixture in the mouse and rat.
N-Acetyl-D-[2-14C,9-3H]neuraminic acid, enzymically prepared from sodium [2-14C]-pyruvate and N-acetyl-D-[6-3H]mannosamine by N-acetylneuraminate lyase in 75% yield, was orally administered to 20 day old fasted mice. 90% of the administered neuraminic acid was absorbed from the intestine in the course of 4 h, at a rate depending on the retention time of neuraminic acid in the intestine and the mental conditions of the animals. Between 60 and 90% of the neuraminic acid was excreted in the urine without chemical alteration within the first 6 h. Four hours after administration 10% of the 3H- and 1.3% of the 14C-radioactivity were recovered in the whole blood and in liver, spleen, kidney and brain. After 3 days 0.5% of 3H- and 0.01% of 14C-radioactivity still remained in these tissues. The discrepancy of the 14C-amount relative to the 3H-quantity was accounted for by exhaled 14CO2. After intravenous injection of N-acetylneuraminic acid into rats, 90% of the radioactivity corresponding to the original substance was excreted in the urine within 10 min. Four hours after administration only 5% of the applied 3H- and 1.2% of the 14C-radioactivity were left in the blood and in liver, spleen, kidney and brain. The experiments show that neither orally nor intravenously applied N-acetylneuraminic acid can penetrate cell membranes to a large extent, with the exception of the intestine. The isotopic ratio and N-acetylneuraminate lyase activity suggest that the small amount of the neuraminic acid retained in tissues was largely cleaved by the lyase, followed by metabolism of the reaction products. It may be concluded from these observations that neuraminic acid occurring in food cannot directly be used for the biosynthesis of glycoconjugates on a large scale.
Animals↗
Phagocytosis of sialidase-treated rat erythrocytes: evidence for a two-step mechanism.
Binding and phagocytosis of rat erythrocytes by liver and peritoneal macrophages were studied with a radioactive in vitro assay which yields quantitative data. Partial removal of sialic acids from the erythrocytes by Vibrio cholerae sialidase resulted in a marked increase of binding of the red cells by both liver and unstimulated peritoneal macrophages. Peritoneal macrophages stimulated by thioglycolate or starch, however, did not differentiate between control and desialylated erythrocytes. By inhibition experiments it was confirmed that rat peritoneal macrophages bind homologous sialidase-treated erythrocytes via a beta-D-galactose-specific lectin on the macrophage surface. While this attachment already occurs in buffer, serum was required for the subsequent phagocytosis. The possible involvement of factors of the complement system in the phagocytosis step was evidenced by a marked decrease of phagocytosis after heat inactivation of the serum. Based on these experiments, we propose a model of a two-step mechanism for the uptake of sialidase-treated erythrocytes by macrophages, comprising both the lectin and a receptor for serum components.
Animals↗
Involvement of membrane galactose in the in vivo and in vitro sequestration of desialylated erythrocytes.
The influence of terminal beta-galactose residues for the in vitro and in vivo sequestration of sialidase-treated erythrocytes by macrophages was investigated. Preincubation of rat peritoneal macrophages with galactose, oligosaccharides, glycoproteins and glycolipids with terminal beta-galactose residues inhibits both binding and phagocytosis of sialidase-treated erythrocytes by masking a beta-galactose-specific lectin on the macrophage cell membrane. These inhibition studies show that binding via demasked erythrocyte surface beta-galactosyl residues to this lectin is necessary for the subsequent phagocytosis step. According to these observations, repeated injections of lactose (30mM serum concentration) and asialo-fetuin (10-30 microM serum concentration) into the blood stream of rabbits led to a reduction of the rapid sequestration rate of sialidase-treated erythrocytes. Asialo-fetuin proved to be a much more potent inhibitor than lactose, in accordance with the in vitro experiments. This inhibition is reversible, as after the disappearance of the inhibitory effect, the sialidase-treated erythrocytes were again rapidly removed from the circulation to an extent similar to that of the experiments without inhibitors. No significant influence on binding and phagocytosis was measured in the presence of sialyllactose and native fetuin in vitro, or of native fetuin on sequestration in vivo. The experiments with rabbits show that a beta-galactosyl-specific lectin seems to be involved in the mechanism of sequestration of desialylated erythrocytes in vivo, as has been observed in vitro with rat peritoneal macrophages.
Animals↗
Purification of a human spermatozoal antigen.
The lack of a more detailed study of spermatozoal antigens lies partly in the difficulty of adequate purification procedures. In the present work a spermatozoal cell membrane antigen was isolated using lithium 3,5-diiodosalicylate as the solubilizing agent. Its apparent molecular weight is 40 500 by gel filtration chromatography and 35 000 by dodecyl sulphate-polyacrylamide gel electrophoresis. Labelling of the antigen with 125I and subsequent radio-immunoprecipitation enabled the evaluation of specific binding to IgG and IgM molecules in sperm-agglutinating and sperm-immobilizing antisera. Adsorption experiments with the purified antigen resulted in significant titer reductions of the same antisera in the microsperm-agglutination and microsperm-immobilization tests. This indicates the presence of an antigen molecule on the human spermatozoa that reacts with IgG and IgM antibodies and with both types of antisera.
Antigen-Antibody Complex↗
Sialidase treatment of rat-erythrocytes: a model for cellular aging?
An in vitro assay system was developed to test the propensity of "old" and sialidase-treated rat erythrocytes to be bound and phagocytosed by rat peritoneal macrophages. Cells considered to be old were phagocytosed to a greater degree than those considered to be young. When erythrocytes were treated with immobilized Vibrio cholerae sialidase, higher amounts of sialic acids had to be removed (25--30%) to induce binding and phagocytosis of the cells, than if the cells had been treated with soluble enzyme (9--15%).
Animals↗
Isolation and characterization of a trisialoganglioside from mouse brain, containing 9-O-acetyl-N-acetylneuraminic acid.
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Animals↗
Identification by gas-liquid chromatography-mass spectrometry of 4-O-acetyl-9-O-lactyl-N-acetyl-neuraminic acid, a new sialic acid from horse submandibular gland.
The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already known equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acid and 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.
Animals↗
Identification of 9-O-acetyl-N-acetylneuraminic acid on the surface of BALB/c mouse erythrocytes.
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Acetylation↗
Metabolism of 4-O-methyl-N-acetylneuraminic acid a synthetic sialic acid.
1. 4-O-Methyl-N-acetylneuraminic acid shows a strong positive periodate-thiobarbiturate reaction. The mechanism of dye formation in this test for sialic acids is discussed in view of the studies already published. 2. An efficient preparation of a tritium-labelled 4-O-methyl-N-acetylneuraminic acid, with high specific radioactivity, by an oxymercuration-demercuration procedure is presented. 3. Sialytransferase activities in microsomal fractions of equine liver using desialylated fetuin are studied. The enzyme activity, assayed in a radioactive procedure, shows an apparent Km value for CMP-N-acetylneuraminic acid of 0.7 mM, whereas this value is 3.4 mM for CMP-4-O-methyl-N-acetylneuraminic acid. Differences are also observed in the maximal velocity for the two substrates. 4. The equine liver system can be used to prepare substantial amounts of fetuin containing radioactive N-acetylneuraminic acid or 4-O-methyl-N-acetylneuraminic acid. The isolated reaction products show similar sialic acid release by treatments with acid or fowl-plague virus neuraminidase. In contrast, 4-O-methyl-N-acetylneuraminic acid-fetuin displays a marked resistance to desialylation by Vibrio cholerae neuraminidase. 5. Free 4-O-methyl-N-acetylneuraminic acid is completely resistant to the action of acylneuraminate pyruvate-lyase. It does not inhibit the enzymic cleavage reaction of N-acetylneuraminic acid. 6. The influence of a substitution at C-4 neuraminic acid on the enzymatic reaction mechanisms is discussed.
Animals↗
Isolation and characterization of acylneuraminate cytidylyltransferase from frog liver.
Frog liver (Rana esculenta) is a rich source of acylneuraminate cytidylyltransferase. The soluble enzyme was purified 250-fold almost to purity with 25% yield and a specific activity of 9 mkat/kg protein (0.54 U/mg protein) using DEAE Sephadex and Sepharose 6B chromatography, followed by preparative polyacrylamide gel electrophoresis. The molecular weight of the cytidylyltransferase was determined to be 163 000 with the aid of Sepharose 6B chromatography and gel electrophoresis, with or without dodecyl sulphate or urea. No subunits were found. The isoelectric point of the enzyme is at pH 6. Optimum reaction rate was observed at pH 9, 37 degrees C, 50mM Mg2 or Ca2 and ImM mercaptoethanol. The Km values for N-acetylneuraminic acid, N-glycoloylneuraminic acid and CTP are 1.6mM, 2.3 mM and 0.6mM, respectively. O-Acetylated sialic acids are inactive with the cytidylyltransferase from frog liver. Enzyme activity can be inhibited by SH reagents and CMP (Ki = 0.5mM).
Animals↗