Interaction of erythrocytes, lymphocytes and thrombocytes with macrophages after treatment with sialidase.
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Animals↗
Biomedical subjects
R Schauer
Publications and source records attributed to R Schauer.
Mechanism of binding and uptake of sialidase-treated blood cells and glycoproteins by the galactose-specific receptor of rat peritoneal macrophages.
Sialidase-treated rat erythrocytes, lymphocytes and thrombocytes bind to homologous peritoneal macrophages via demasked, terminal galactose residues on the blood cells and a galactose-specific receptor on macrophages. While erythrocytes and thrombocytes are phagocytosed after binding, vital lymphocytes are released from the phagocytes after several hours of incubation, because their surface sialic acids are resynthesized. Asialoglycoprotein-coated gold-particles are also bound, and the pathway of their internalization and recycling of the receptor was studied using various inhibitors of endocytosis. Sialidase treatment of macrophages abolished their binding capacity.
Animals↗
[Cervicomotography].
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Cervical Vertebrae↗
Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver.
The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.
Animals↗
Transport of CMP-N-glycoloylneuraminic acid into mouse liver Golgi vesicles.
CMP-Neu5Gc has been shown to be transported into mouse liver Golgi vesicles by a specific carrier the characteristics of which were investigated in detail. In the system employed, CMP-Neu5Gc enters the Golgi vesicles within 2 min; transport was saturable with high concentrations of the sugar-nucleotide and was dependent on temperature. A kinetic analysis gave an apparent Km of 1.3 microM and a maximal transport velocity of 335 pmol/mg protein per min. Almost identical values were obtained with CMP-Neu5Ac, under the same incubation conditions. Furthermore, the uptake of CMP-Neu5Gc was inhibited by CMP-Neu5Ac, a substrate analogue. Conversely, the uptake of CMP-Neu5Ac was inhibited by CMP-Neu5Gc to the same extent, leading to the conclusion that the transport of CMP-Neu5Ac and CMP-Neu5Gc is mediated by the same carrier molecule. This transport system for CMP-Neu5Gc involves both CMP and CMP-Neu5Gc since intravesicular CMP induced the entry of external CMP-Neu5Gc.
Animals↗
Conserved sequences in bacterial and viral sialidases.
The genes of the bacterial sialidases from Clostridium sordellii G12, C. perfringens A99, Salmonella typhimurium LT-2 and Vibrio cholerae 395 sequenced so far were examined for homologies and were compared with sequences of viral sialidases. Each of the bacterial sialidases contains a short sequence of twelve amino-acids, which is repeated at four positions in the protein. All these sequences exhibit significant similarities. Comparing the repeated sequences of the four sialidases, five amino-acids were found to be highly conserved at defined positions: Ser-X-Asp-X-Gly-X-Thr-Trp. Additionally, most of the distances between the four repeated regions are also conserved among the different sialidases. The conserved bacterial sequences show similarity with sialidases of influenza A H7N1 and H13N9.
Amino Acid Sequence↗
A detailed study of the periodate oxidation of sialic acids in glycoproteins.
Periodate oxidation of terminal N-acetyl- and N-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions, N-glycoloylneuraminic acid is oxidized about 1.5 times faster than the N-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz 1H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe3+/HCl assay were determined.
Animals↗
Partial purification and characterization of sialate O-acetylesterase from bovine brain.
From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylesterase↗
Cloning, sequencing and expression of a sialidase gene from Clostridium sordellii G12.
A 4.3 kb XbaI restriction fragment of DNA from Clostridium sordellii G12 hybridized with a synthetic oligonucleotide representing the N-terminus of the sialidase protein secreted by C. sordellii. This cloned fragment was shown to encode only part of the sialidase protein. The sialidase gene of C. sordellii was completed by a 0.7 kb RsaI restriction fragment overlapping one end of the XbaI fragment. After combining the two fragments and transformation of Escherichia coli, a clone that expressed sialidase was obtained. The nucleotide sequence of the sialidase gene of C. sordellii G12 was determined. The sequence of the 18 N-terminal amino acids of the purified extracellular enzyme perfectly matched the predicted amino acid sequence near the beginning of the structural gene. The amino acid sequence derived from the complete gene corresponds to a protein with a molecular mass of 44,735 Da. Upstream from the putative ATG initiation codon, ribosomal-binding site and promoter-like consensus sequences were found. The encoded protein has a leader sequence of 27 amino acids. The enzyme expressed in E. coli has similar properties to the enzyme isolated from C. sordellii, except for small differences in size and isoelectric point. Significant homology (70%) was found with a sialidase gene from C. perfringens.
Amino Acid Sequence↗
Isolation and characterization of a sialidase from the starfish Asterias rubens.
The starfish Asterias rubens contains a soluble sialidase (1.4 mU/mg homogenate protein), which was purified over 500-fold to apparent homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography on immobilized 2-deoxy-2,3-didehydroneuraminic acid. The native sialidase has a molecular mass of 230 kDa (gel filtration) and consists of 4 subunits of each 63 kDa, as determined by SDS-gel electrophoresis. Its isoelectric point is at pH 4.9, the activity is optimum at pH 4.2 and 37 degrees C, and it hydrolyses preferably 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acid, followed by sialyllactose and glycoproteins. The hydrolysis rate is decreased or stopped by the presence of O-acetyl groups on the sialic-acid residue to be cleaved. N-Glycoloyl residues also retard enzyme action, as well as alpha(2-6) bonds when compared with alpha(2-3) linkages. This relatively stable enzyme is inhibited by mercury or copper ions, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and by the increase of ionic strength. The evolutionary significance of starfish sialidase is discussed.
Animals↗
Properties of sialidase isolated from Actinomyces viscosus DSM 43798.
The cell-bound sialidase of Actinomyces viscosus DSM 43798 was solubilized by mechanical cell disruption and lysozyme treatment. The enzyme was enriched 30,000-fold by cation-exchange chromatography, gel-filtration, and FPLC ion-exchange chromatography, thus obtaining 10 micrograms sialidase protein from 26 g wet cells with a specific activity of 680 U/mg protein. Since sialidase activity was also found in the culture medium, this enzyme was isolated as well, requiring the additional application of FPLC gel-filtration. Both sialidase preparations were apparently homogenous on SDS-PAGE and have similar properties. The substrate specificity of the A. viscosus sialidase was tested with 16 sialoglycoconjugates: The enzyme showed a higher activity with serum glycoproteins than with gangliosides, mucins or sialyllactoses. 4-O-Acetylated N-acetylneuraminic acid was not cleaved from equine submandibular gland mucins or serum glycoproteins in contrast to N-acetyl- and N-glycoloylneuraminic acid. 9-O-Acetyl-N-acetylneuraminic acid was released from bovine submandibular gland mucin, as confirmed by TLC. The sialidase hydrolyses alpha(2----6)-linkages more rapidly than alpha(2----8)- and alpha(2----3)-bonds. Cations, except Hg2+, or chelating agents have no influence on enzyme activity. The sialidase has a relatively high molecular mass of 150 kDa, but consists of only one unit. The enzyme is labile towards freezing and thawing, but can be stored at 4 degrees C in 0.1 M acetate buffer, pH 5.
Actinomyces↗
Cloning and sequencing of a Clostridium perfringens sialidase gene.
Escherichia coli was transformed with pUC vectors containing Sau3A restriction fragments (RF) of Clostridium perfringens DNA. Two clones expressed sialidase activity when assayed with the fluorogenic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid. A synthetic oligonucleotide representing the N-terminus of the expressed enzyme hybridized with the clostridial insert and with a corresponding 2.1 kb Sau3A RF of the C. perfringens genome. The insert reduced to 1.4 kb, which still encoded active sialidase, has been sequenced. The structural gene encodes 382 amino acids representing an Mr of 42770. A hydrophobic leader sequence is absent. Upstream from the initiation codon ATG, a GA-rich region is found and considered as the Shine-Dalgarno sequence. Homology with the N-terminus of the Vibrio cholerae sialidase gene and with viral sialidase sequences was not found.
Amino Acid Sequence↗
Early diagnosis of clostridial gas gangrene using sialidase antibodies.
In order to improve the diagnosis of gas gangrene, especially at an early stage of infection, new ways for the detection of the responsible Clostridia were investigated. Sialidase, known to be excreted in large amounts by the most frequently occurring myonecrotizing clostridial species, Clostridium perfringens, Clostridium septicum, and Clostridium sordellii, was isolated. With polyclonal antibodies raised against these enzymes, two immunological assays were established, which are directed against the sialidase activity (sialidase inhibition test) and the enzyme protein ('sandwich'-ELISA), respectively. Using these assays, species-specific information about the presence of clostridial sialidase was obtained within 50 min or 6 h. Animal tests revealed that both assays are applicable 8-12 h after clostridial infection, using resected tissues or wound fluids for estimations. The assays allow specific, sensitive, and quantitative measurement of clostridial sialidases, and no significant interference by sialidases from other microbes or from host tissues occurred. The applicability of the new assays for an early diagnosis of gas gangrene in human patients is discussed.
Animals↗
Sialic acids as antigenic determinants of complex carbohydrates.
Sialic acids, comprising a group of N- and O-acyl as well as O-methyl and O-sulfate derivatives of neuraminic acid, are differently linked to various sugars of glycoproteins and gangliosides. Thus, the great molecular variety already existing in the asialo-portion of glycoconjugates is much increased by the addition of sialic acids. This structural diversity may be mirrored by a high antigenic diversity of the oligosaccharide part of complex carbohydrates. However, not much is known of such an influence of the sialic acid moieties of glycane chains, although antibodies against many non-sialylated oligosaccharides are known. In most cases investigated so far, sialic acids reduce or even prevent antigenicity of oligosaccharide chains and of the protein or lipid parts of glycoconjugate molecules, respectively. Enzymic removal of sialic acid or reduction of its carboxylic group to an alcohol residue alters or significantly increases the antigenicity. In contrast to this masking effect, sialic acids have been found to act as antigenic determinants. N-Acetylneuraminic acid in different linkages to other sugars is an essential component of various antigens in human erythrocytes, and N-glycolylneuraminic acid determines the blood group specificity of East-Asian dogs. The latter sialic acid also plays a role in immunological processes in man. N-Glycolylneuraminic acid probably cannot be synthesized in man, but is derived from the foodstuffs and incorporated into tissue glycoconjugates in small quantities. Sialic acid O-acetyl groups are also involved in immunological processes by modifying the antigenicity of glycoproteins and polysaccharides or by contributing to the specificity of tumor antigens. Evidence for a role of the multiple forms of sialic acids as differentiation antigens is accumulating.
Animals↗
Sialate O-acetylesterases: key enzymes in sialic acid catabolism.
Sialate 9(4)-O-acetylesterases (EC 3.1.1.53) have been isolated from equine liver, bovine brain and influenza C virus. In this latter case, the esterase represents the receptor-destroying enzyme of the virus. The kinetic properties of these enzymes were determined with Neu5,9Ac2 and in part with 4-methylumbelliferyl acetate and Neu5,9Ac2-lactose. The Km values vary between 0.13 and 24 mM and the Vmax values from 0.55 to 11 U/mg of protein. The pH optima are in the range of 7.4-8.5, the molecular masses at 56,500 and 88,000 Da. In addition to a fast hydrolysis found for aromatic acetates, such as 4-methylumbelliferyl acetate or 4-nitrophenyl acetate, N-acetyl-9-O-acetylneuraminic acid is de-O-acetylated at the highest relative rate. Other substituents at the 9-position, such as lactoyl residues, or acetyl groups at other positions within the side chain are not hydrolyzed. Neu4,5Ac2, however, is a substrate for all 3 enzymes. The hydrolysis rates of this ester function, which renders sialic acids resistant to the action of sialidases, vary from 3 to 100% relative to Neu5,9Ac2. Whereas Neu5,9Ac2-lactose is hydrolyzed by the bovine and viral esterases, other O-acetylated sialic acids in glycoconjugates are only attacked by the enzyme from influenza C virus and not by that from bovine brain. The esterase from horse liver also releases 4-O-acetyl groups from equine submandibular gland mucin. By incubation with appropriate substrates and inhibition studies, carboxylesterase, amidase and choline esterase activities were excluded, as well as the cleavage of other acyls, e.g., butyryl groups. Thus, the enzymes investigated belong to the acetylesterases.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylesterase↗
The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid.
The biosynthesis of N-glycoloylneuraminic acid in fractionated porcine submandibular glands was investigated. The following substrates: [3H]N-acetylmannosamine, free [14C]N-acetylneuraminic acid, CMP-[14C]N-acetylneuraminic acid, [14C]N-acetylneuraminic acid linked alpha(2----3) to galactose residues, or alpha(2----6) to Gal-beta(1----4)-GlcNAc residues of porcine submandibular mucin and [14C]N-acetylneuraminic acid linked alpha(2----6) to GalNAc residues of ovine submandibular gland mucin were incubated, in the presence of cofactors, with the soluble protein, heavy membrane and microsomal fractions of porcine submandibular glands. Radio thin-layer chromatographic analysis revealed that only one substrate, CMP-[14C]N-acetylneuraminic acid, was hydroxylated. The product was identified as CMP-[14C]N-glycoloylneuraminic acid by (i) co-chromatography with non-radioactive CMP-N-glycoloylneuraminic acid standard, (ii) acid hydrolysis to free [14C]N-glycoloylneuraminic acid, (iii) alkaline hydrolysis to yield N-glycoloylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid and (iv) transfer of [14C]N-glycoloylneuraminic acid to asialo-fetuin by sialyltransferase. 85% of CMP-N-acetylneuraminic acid hydroxylase activity was present in the soluble protein fraction, with small amounts of activity in the two particulate fractions. The CMP-N-acetylneuraminic acid hydroxylase in the soluble protein fraction had an absolute requirement for Fe2+ ions and a reducing cofactor. NADPH and NADH were by far the most effective cofactors, smaller amounts of hydroxylation could, however, be supported by ascorbic acid and 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin.
Animals↗
Isolation and characterization of sialate 9(4)-O-acetylesterase from influenza C virus.
An esterase was isolated from influenza C virus with a specific activity from 1.7-5 U/mg protein, and its substrate specificity was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. The enzyme hydrolyses only acetic acid esters at significant rates. The non-natural substrates 4-methyl-umbelliferyl acetate, 4-nitrophenyl acetate, and alpha-naphthyl acetate are cleaved at highest hydrolysis rates, followed by the natural substrate N-acetyl-9-O-acetylneuraminic acid. The esterase also acts on N-glycoloyl-9-O-acetylneuraminic acid and, much slower, on N-acetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid is not hydrolysed. 2-Deoxy-2,3-didehydro-N-acetyl-9-O-acetylneuraminic acid is also a substrate for this enzyme, however, 6-O-acetylated N-acetylmannosamine and glucose are not. Esterification of the carboxyl function of sialic acids strongly reduces or prevents esterase action on O-acetyl groups. The carboxyl ester is not hydrolysed. The relative cleavage rates also depend on the type of the non-sialic acid part of the molecule. N-Acetyl-9-O-acetylneuraminic acid as component of sialyllactose and rat serum glycoprotein shows hydrolysis rates close to the free form of this sugar, while acetyl ester groups of bovine submandibular gland mucin and rat erythrocytes are hydrolysed at slower rates. Gangliosides and 4-O-acetylated glycoproteins are no substrates for the purified enzyme. A slow hydrolysis is observed by incubation of 9-O-acetylated GD1a with intact influenza C viruses. As other natural acetyl esters (acetyl-CoA and acetylthiocholine iodide) are not hydrolysed, the enzyme can be classified as sialate 9(4)-O-acetylesterase (EC 3.1.1.53).
Acetylesterase↗
The galactose-recognizing system of rat peritoneal macrophages; identification and characterization of the receptor molecule.
Resident rat peritoneal macrophages express a galactose-recognizing system, which mediates binding and uptake of cells and glycoproteins exposing terminal galactose residues. Here we describe the identification, isolation, and characterization of the corresponding receptor molecule. Using photoaffinity labelling of adherent peritoneal macrophages with the 4-azido-6-125I-salicylic acid derivative of anti-freeze glycoprotein 8 followed by SDS-PAGE and autoradiography, we identified the receptor of these cells as a protein with an apparent molecular mass of 42 kDa. Furthermore, cell surface receptors were radioiodinated by an affinity-supported labelling technique using the conjugate of asialoorosomucoid and lactoperoxidase, followed by extraction and isolation by affinity chromatography. Finally, the native receptor was isolated and analysed. To estimate its binding activity in solutions, a suitable binding assay was developed, using the precipitation of receptor-ligand complex with polyethylene glycol to separate bound from unbound 125I-asialoorosomucoid, which was used as ligand. It is shown that the isolated receptor binds to galactose-exposing particles and distinguishes between sialidase-treated and -untreated erythrocytes, similar to peritoneal macrophages. The binding characteristics of the membrane-bound and the solubilized receptor are described in the following paper of Lee et al.
Animals↗