Glycosylation patterns of mucins in colonic disease.
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Acetylation↗
Biomedical subjects
R Schauer
Publications and source records attributed to R Schauer.
Antibody-mediated imbalance of myocardial energy metabolism. A causal factor of cardiac failure?
The ADP-ATP carrier of the inner mitochondrial membrane is an autoantigen in myocarditis and dilated cardiomyopathy. Sera of patients with these diseases contain carrier-specific autoantibodies that inhibit the transmembrane nucleotide transport on isolated mitochondria. Guinea pigs immunized with the isolated ADP-ATP carrier protein also generate specific carrier-inactivating antibodies. In this study, we measured the cardiac function of guinea pigs immunized with the ADP-ATP carrier by determining the external heart work (EHW) of their isolated perfused spontaneously beating hearts stimulated by 4.0 mmol/L calcium and aortic ligature. Further, the electrogenic transport activity of the ADP-ATP carrier was estimated by calculating the cytosolic-mitochondrial difference of the phosphorylation potential of ATP [delta G(cyt-mit)] in the freeze-clamped isolated hearts by nonaqueous fractionation. The EHW of immunized guinea pigs was seen to be reduced by 54% (P < .005) compared with nonimmunized control guinea pigs, and delta G(cyt-mit) declined from 4.9 kJ/mol ATP in nonimmunized control hearts to 2.3 kJ/mol ATP in the hearts of the immunized guinea pigs (P < .005). The decisive result of this study, however, is the close relation observed between the magnitude of reduction of delta G(cyt-mit) and the size of the decrease in EHW (r = .87). Therefore, it seems plausible that antibody-mediated carrier dysfunction (creating the observed imbalance in myocardial energy metabolism) is responsible for the impairment of cardiac function. Our data support the hypothesis that immunopathic mechanisms in myocarditis and dilated cardiomyopathy can trigger subsequent heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate↗
Diversity in the properties of two sialidase isoenzymes produced by Clostridium perfringens spp.
Clostridium perfringens produces two sialidases, one of which has a molecular mass of 71 kDa and is secreted, while the 'small', 43 kDa isoenzyme remains in the cells. The secreted, higher molecular mass sialidases of two different clostridial strains, DSM756T and A99, exhibit maximum activity at pH 5.5 and at 51 or 55 degrees C, respectively. The molecular mass of both enzymes is 71 kDa in SDS-PAGE and 63 kDa as determined by gel-filtration, which indicates the absence of subunits. Natural sialidase substrates are hydrolyzed at comparably high rates, e.g. the glycoproteins fetuin and bovine submandibular gland mucin, the homopolymer colominic acid, and the ganglioside mixture from bovine brain. The partially purified 'small' isoenzyme from C. perfringens A99 cells had similar properties to the corresponding recombinant sialidase isolated from the Escherichia coli host. It is located inside the clostridial and E. coli cells and exhibits maximum activity at pH 6.1 and 37 degrees C. A relative molecular mass of 32,000 was found with FPLC gel-filtration chromatography, while primary structure analysis yielded a value of 43,000. It differs a significantly from the 'large' isoenzyme by substrate specificity. Preferred substrates are oligosaccharides, while other, more complex sialoglycoconjugates are hydrolyzed only at very low rates. alpha 2,3-linkages are hydrolyzed much faster than alpha 2,6-bonds.
Chromatography, Gel↗
Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily.
BACKGROUND: Protein-carbohydrate interactions are believed to be important in many biological processes that involve cell-cell communication. Apart from the selectins, the only well-characterized vertebrate sialic acid-dependent adhesion molecules are CD22 and sialoadhesin; CD22 is a member of the immunoglobulin superfamily that is expressed by B lymphocytes and sialoadhesin is a macrophage receptor. The recent cloning of the gene encoding sialoadhesin has shown that it is also immunoglobulin-like. Both proteins share sequence similarity with the myelin-associated glycoprotein, an adhesion molecule of oligodendrocytes and Schwann cells that has been implicated in the process of myelination, raising the important question of whether myelin-associated glycoprotein is also a sialic acid-binding protein. RESULTS: We have investigated the binding properties of these three receptors when expressed either in monkey COS cells or as chimaeric proteins containing the Fc portion of human immunoglobulin G. We demonstrate that, like sialoadhesin and CD22, myelin-associated glycoprotein mediates cell adhesion by binding to cell-surface glycans that contain sialic acid. We have dissected the specificities of these three adhesins further: whereas sialoadhesin binds equally to the sugar moieties NeuAc alpha 2-->3Gal beta 1-->3(4)GlcNAc or NeuAc alpha 2-->3Gal beta 1-->3GalNAc, myelin-associated glycoprotein recognizes only NeuAc alpha 2-->3Gal beta 1-->3GalNAc and CD22 binds specifically to NeuAc alpha 2-->6Gal beta 1-->4GlcNAc. Furthermore, we show that the recognition of sialylated glycans on the surfaces of particular cell types leads to the selective binding of sialoadhesin to neutrophils, myelin-associated glycoprotein to neurons and CD22 to lymphocytes. CONCLUSIONS: Our findings demonstrate that a subgroup of the immunoglobulin superfamily can mediate diverse biological processes through recognition of specific sialylated glycans on cell surfaces. We propose that this subgroup of proteins be called the sialoadhesin family.
Animals↗
CMP-N-acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands. Interaction with membrane-bound and soluble cytochrome b5-dependent electron transport chains.
In this report, the nature of the protein components involved in the functioning of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5 Ac) hydroxylase in high-speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH-cytochrome b5 reductase, cytochrome b5 and a 56-kDa terminal electron acceptor having the CMP-Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction; however, the amphipathic, usually membrane-associated, forms of cytochrome b5 and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylase in vivo. Although the majority of cellular cytochrome b5 and cytochrome b5 reductase is membrane-bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent-solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochrome b5. Accordingly, in reconstitution experiments, pure hydrophilic cytochrome b5 interacts more effectively with the hydroxylase than isolated amphipathic cytochrome b5. Studies on the CMP-Neu5 Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containing N-glycolylneuraminic acid.
Animals↗
Gene structure of the 'large' sialidase isoenzyme from Clostridium perfringens A99 and its relationship with other clostridial nanH proteins.
Clostridium perfringens possesses two sialidase isoenzymes of different molecular weight. Almost 90% of the gene encoding the 'large' form was found on a 3.1 kb chromosomal fragment (Sau3AI) of strain A99 by hybridization with probes developed from the N-terminal protein sequence and from commonly conserved sialidase motifs ('Asp-boxes'), whereas the remaining 3'-terminal part was detected on a 2.1 kb fragment (Hind III) of chromosomal DNA. After combination of both fragments, the resulting E. coli clones expressed sialidase activity, the properties of the recombinant sialidase corresponding with those of the wild type enzyme. The entire chromosomal fragment of 3665 bp encompasses the complete sialidase gene of 2082 bp corresponding to 694 amino acids, from which a molecular weight of 72,956 for the mature protein can be deduced. The first 41 amino acids are mostly hydrophobic and probably represent a signal peptide. The sialidase structural gene follows a non-coding region with an inverted repeat and a ribosome-binding site. Upstream from the regulatory region, another open reading frame (ORF) was detected. The 3'-terminus of the sialidase structural gene is directly followed by a further ORF of unknown function, which possibly encodes a putative permease or the acylneuraminate pyruvate-lyase involved in sialic acid catabolism. The primary structure of the 'large' isoenzyme is very similar to the sialidase of Clostridium septicum (55% identical amino acids), whereas the homology with the 'small' form of the same species is comparatively low (26%).
Amino Acid Sequence↗
Purification, characterization and reconstitution of CMP-N-acetylneuraminate hydroxylase from mouse liver.
CMP-N-acetylneuraminate hydroxylase was isolated from mouse liver high speed supernatant with a yield of 0.4% and an apparent 1000-fold purification. The enzyme is a monomeric protein with a molecular weight of 66 kDa, as determined by gel filtration and SDS-PAGE. The hydroxylase system was reconstituted with Triton X-100-solubilized mouse liver microsomes and purified soluble or microsomal forms of cytochrome b5 reductase and cytochrome b5. The systems were characterized in detail and kinetic parameters for each system were determined.
Animals↗
Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22.
An increasing number of mammalian cell adhesion molecules, including sialoadhesion, CD22 and the family of selectins, have been found to bind cell surface glycoconjugates containing sialic acids. Here we describe how the structural diversity of this sugar influences cell adhesion mediated by the related molecules sialoadhesin and CD22 in murine macrophages and B-cells respectively. We show that the 9-O-acetyl group of Neu5,9Ac2 and the N-glycoloyl residue of Neu5Gc interfere with sialoadhesin binding. In contrast, CD22 binds more strongly to Neu5Gc compared to Neu5Ac. Of two synthetic sialic acids tested, only CD22 bound the N-formyl derivative, whereas a N-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.
Acetylation↗
Determination of sialic acids.
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Animals↗
Trans-sialidases in the insect-vector stages of African and American trypanosomes: reply.
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Comment↗
Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus.
Influenza C virus spike glycoprotein HEF specifically recognizes glycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid. The same protein also contains an esterase activity. Taking advantage of these two properties, influenza C virus was used as a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminic acid in human leucocytes. The binding of influenza C virus to leucocyte glycoproteins and gangliosides separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and thin-layer chromatography, respectively, was assayed using a chromogenic esterase substrate. In this way, glycoproteins of B-lymphocytes and T-lymphocytes were found to contain 9-O-acetylated sialic acids. Of the various 9-O-acetylated gangliosides detected, one had the characteristics of 9-O-acetylated GD3. The identification of 9-O-acetylated sialic acids on distinct glycoproteins and glycolipids should be helpful in assigning a physiological role to this sugar.
Acetylation↗
Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands.
N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-glycoside by the action of CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase. This enzyme is a soluble cytochrome b5-dependent monooxygenase and has been purified to apparent homogeneity from pig submandibular glands by precipitation with N-cetyl-N,N,N-trimethylammonium bromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose, Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12. This procedure resulted in an 8960-fold purification of the hydroxylase with a recovery of 0.8%. The molecular mass of this protein was shown to be 65 kDa on SDS-PAGE and approximately 60 kDa as determined by gel filtration on Superose S.12, which suggests that the enzyme is a monomer. The purified CMP-Neu5Ac hydroxylase is activated by FeSO4 and inhibited by iron-binding reagents such as o-phenanthroline, KCN, Tiron and ferrozine. An apparent Km of 11 microM was determined for the substrate CMP-Neu5Ac using purified hydroxylase in the presence of Triton X-100-solubilized microsomes. In a reconstituted system consisting of purified hydroxylase, cytochrome b5, cytochrome b5 reductase and catalase, an apparent Km of 3 microM was measured. The apparent Km for cytochrome b5 in this system was 0.24 microM. Immunization of a rabbit with enriched and purified hydroxylase led to an antiserum that inhibited CMP-Neu5Ac hydroxylase activity and reacted with the purified 65 kDa protein on a Western blot after SDS-PAGE. Antibodies specific for this 65 kDa protein were isolated and showed a strong reaction with the purified CMP-Neu5Ac hydroxylase from mouse liver after immunoblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Catalytic properties of the CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens: comparison with the mammalian enzyme.
The biosynthesis of N-glycolylneuraminic acid (Neu5Gc) was investigated in cell-free extracts of the starfish Asterias rubens, which is one of the evolutionarily least-advanced species known to possess Neu5Gc-containing glycoconjugates. As in higher animals, Neu5Gc is synthesised in Asterias rubens by the action of a CMP-Neu5Ac hydroxylase. Enzyme activity was detected in all starfish tissues tested, the turnover being the greatest in the gonads. The enzyme from this tissue has a temperature optimum between 25 and 33 degrees C and a pH optimum between pH 6.0 and 6.4. This hydroxylase exhibits many characteristics in common with the mammalian enzyme. For example, the enzyme is extracted in a predominantly soluble form. Oxygen and a reduced pyridine nucleotide are necessary for activity, with NADH being the most effective cofactor. Furthermore, the activation of the hydroxylase by exogenously added iron salts and the potent inhibitory effects of several iron ligands point to the involvement of a non-haem iron cofactor. The enzyme has a high affinity for the substrate CMP-Neu5Ac, the apparent Km being 18 microM. In contrast to the mammalian enzyme, the hydroxylase from Asterias rubens is not inhibited by increased ionic strength and cannot be activated by non-ionic detergents. Moreover, the CMP-Neu5Ac turnover increased linearily with increasing protein concentration. In accordance with other enzymes in starfish, seasonal changes in the CMP-Neu5Ac hydroxylase activity were also observed.
Animals↗
An immunoassay for the rapid and specific detection of three sialidase-producing clostridia causing gas gangrene.
A rapid and methodologically unusual diagnostic test was developed for the specific detection of Clostridium perfringens, C. septicum and C. sordellii, which cause clostridial myonecrosis. Sialidases (EC 3.2.1.18) secreted by these bacterial species were bound to polyclonal antibodies raised against the respective enzyme and immobilized onto microtiter plates. The activity of bound sialidase was determined with the fluorogenic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid. The assay permits the detection of a minimum sialidase activity of about 0.1-1 mU/ml of sample solution within 2 h. The sensitivity of the test was reduced by about three-fold for sialidase activities in samples containing 50% serum. Only a few, low cross-reactivities, which never exceeded 10% of the homologous reaction, were observed with 12 sialidases from other bacterial sources. Clinical isolates of the three clostridial species were analysed by this assay and gave positive signals in the homologous test. The assay for the detection of C. perfringens was applied to nine samples from patients suspected to be suffering from clostridial myonecrosis. There was a high correlation between the results of the immunoassay and the bacteriological analysis of infection.
Animals↗
Isolation and properties of the natural and the recombinant sialidase from Clostridium septicum NC 0054714.
The natural sialidase of Clostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed by Escherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 degrees C in 60 mM sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having alpha(2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the alpha(2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. alpha(2-3) Linkages are more rapidly hydrolysed than alpha(2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.
Cloning, Molecular↗
Indications for the enzymatic synthesis of 9-O-lactoyl-N-acetylneuraminic acid in equine liver.
Fractionation of horse liver homogenate by centrifugation into heavy membranes at 10,000 x g, microsomal fraction at 105,000 x g, and the supernatant revealed sialate 9-O-lactoyltransferase activity only in the latter fraction. For the enzyme assay, the various fractions were incubated with 14C labelled CMP-N-acetylneuraminic acid, N-acetylneuramimic acid and glycoconjugate-bound N-acetylneuramimic acid. Lactoylation was identified in three different TLC systems after acid hydrolysis and purification of the sialic acids in the incubation mixtures. Enzyme activity was found only in the supernatant fraction. Glycoconjugate-bound N-acetylneuramimic acid was the best substrate tested, although some lactoylation was also found when using CMP-N-acetylneuraminic acid.
Acyltransferases↗
Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes.
The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides GM3 (II3Neu5Ac-LacCer), neolacto-series gangliosides (IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer) containing C24:1, and to some extent C22:0; and C16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Ac alpha 2-3Gal and/or Neu5Ac alpha 2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions.
Carbohydrate Sequence↗
The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein.
A developmentally regulated trans-sialidase activity is present on the surface of procyclic Trypanosoma brucei. Bloodstream stages display no trans-sialidase activity. T. brucei trans-sialidase is capable of transferring sialic acids from a variety of glycoconjugates into new glycosidic linkages without requirement for CMP-Neu5Ac. The enzyme is linked to the plasma-membrane via a GPI-PLC-resistant GPI-anchor. The comparison of enzymic and structural features of sialidase and trans-sialidase suggests that the two activities may be catalyzed by the same protein, since highly enriched sialidase fractions display trans-sialidase activity. 2-Deoxy-2,3-didehydro-N-acetylneuraminic acid is only a poor inhibitor for the two enzymic activities. Sialic acids are transferred to alpha (2-3)-positions of terminal beta-galactose residues of oligosaccharides and glycoconjugates at various rates. Neu5Ac-alpha(2-3)-lactose is the best trans-sialylation donor tested. Lewis is a poor sialic acid acceptor. T. brucei trans-sialidase utilizes serum glycoconjugates, human and bovine erythrocytes as sialic acid donors, and resialylates sialidase-treated erythrocytes. The enzyme transfers sialic acids from the GPI-anchor of procyclic acidic repetitive protein (PARP) onto lactose and vice versa. Also structures within a variant surface glycoprotein (sVSG MITat. 1.7.) can be trans-sialylated.
Animals↗