共查询到20条相似文献,搜索用时 109 毫秒
1.
Roland Schauer Sabine Stoll Erich Zbiral Erwin Schreiner HanNelore H Brandstetter Andrea Vasella Franz Baumberger 《Glycoconjugate journal》1987,4(4):361-369
Various deoxy- and epi-derivatives ofN-acetylneuraminic acid were synthesized and tested for their substrate properties withN-acetylneuraminate lyase fromClostridium perfringens.N-Acetyl-9-deoxyneuraminic acid is a good substrate,N-acetylneuraminic acid derivatives with epimeric configuration at C-7, C-8 or both are cleaved slowly, whileN-acetyl-4-epi-,N-acetyl-4-deoxy-,N-acetyl-7-deoxy-andN-acetyl-8-deoxyneuraminic acid are resistant to enzyme action.N-Acetyl-4-deoxyneuraminic acid andN-acetyl-4-epineuraminic acid competitively inhibit the enzyme. These studies give further insight into a mechanism proposed for the reversible cleavage of sialic acids byN-acetylneuraminate lyase. 相似文献
2.
Roland Schauer Jorge Casals-stenzel Anthony P Corfield Rüdiger W Veh 《Glycoconjugate journal》1988,5(3):257-270
Bovine submandibular glands were homogenized and fractionated under conditions which yielded subcellular fragments from mainly one cell type, the mucous acinar cell, as judged by morphological analysis of the glands before and after homogenization. The majorN-acetylneuraminate-9(7)-O-acetyltransferase activity was detected in the cytosolic fraction, a result supported by the high specific radioactivity of free sialic acids isolated after [14C]acetate-labelling experiments. Separation of membranes on a Ficoll density gradient gave six fractions which were analyzed biochemically and morphologically. The particulate activities of acetyltransferase and sialyltransferase were found in fractions containing smooth and mitochondrial membranes. MembraneO-acetyl sialic acids were present at the highest levels in these fractions and also had the highest specific radioactivity after [14C]acetate-labelling experiments. Significant amounts of theO-acetyltransferase activity also occur in the cytosol and are consistent with a model ofO-acetyl sialic acid biosynthesis involving both cytosolic and smooth membrane sites ofO-acetylation. 相似文献
3.
Anthony P. Corfield Clarice Do Amaral Corfield Rüdiger W. Veh Susan A. Wagner John R. Clamp Roland Schauer 《Glycoconjugate journal》1991,8(4):330-339
Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio ofN-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin.Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts ofN-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content ofN-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8×105 Da and aggregates in excess of 106 Da, while the minor mucin ranged from 3.0 × 105 to 106 Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.Abbreviations PBS
0.01m sodium phosphate buffer, pH 7.3, containing 0.15m NaCl
- Neu5Ac
N-acetylneuraminic acid
- Neu5Gc
N-glycoloylneuraminic acid
- GalNAc-ol
N-acetylgalactosaminitol 相似文献
4.
T. J. Budd C. D. Dolman A. M. Lawson W. Chai J. Saxton F. W. Hemming 《Glycoconjugate journal》1992,9(5):274-278
N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac
N-acetylneuraminic acid
- Neu5Gc
N-glycoloylneuraminic acid
- RPM
rat promegakaryoblast
- MEG-01
human megakaryoblastic leukaemia cell line
- PAD
pulsed amperometric detection
- WGA
wheat germ agglutinin
- FCS
foetal calf serum
- PPEADF
phosphatidylethanolamine dipalmitoyl
- LSIMS
liquid secondary ion mass spectrometry
- HPAEC
high performance anion exchange chromatography
- TBA
thiobarbituric acid 相似文献
5.
Siiri Hirmo Sørge Kelm Roland Schauer Bo Nilsson Torkel Wadström 《Glycoconjugate journal》1996,13(6):1005-1011
Helicobacter pylori is a human pathogen associated with gastritis and peptic ulcer. Adhesion properties ofH. pylori to various structures have been described in the literature, including evidence for sialic acid-binding. To study the specificity and frequency of sialic acid-binding, fourteenH. pylori strains were investigated using haemagglutination with derivatized erythrocytes carrying sialic acids only on defined glycans and using haemagglutination inhibition assays. From these studiesH. pylori strains can be grouped into sialic acid-dependent and sialic acid-independent classes. The sialic acid-dependent strains require -2,3-linked sialic acid for haemagglutination. The potential roles of sialic acid-dependent adhesions forH. pylori-related infections are discussed.Abbreviations Sia
sialic acids
- Neu5Ac
N-acetyl-neuraminic acid
- Neu5Gc
N-glycolylneuraminic acid
- Neu5Fm
N-formylneuraminic acid
- Neu5TFA
N-trifluoroacetylneuraminic acid
- RBC
human red blood cells (erythrocytes) 相似文献
6.
Rodrigues ML Dobroff AS Couceiro JN Alviano CS Schauer R Travassos LR 《Glycoconjugate journal》2002,19(3):165-173
Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an 2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-14C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat 2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003. 相似文献
7.
Kositprapa C Zhang B Berger S Canty JM Lee TC 《Molecular and cellular biochemistry》2000,215(1-2):47-55
A 32 kDa estrogen-induced, sialic acid-specific agglutinin (P-SAS) was isolated from rat endometrium in its proestrus stage [1]. To investigate the functional importance of P-SAS in the uterine milieu, specific binding assays were carried out with 125I-labeled P-SAS and different cellular components of the uterus (epithelial, stromal and myometrial cells), that were isolated from different stages of the estrus cycle. The results indicate that although the protein is secreted from the epithelial cells in the estrogenic phase, it binds specifically to the stromal cells, especially to those isolated from the diestrus stage of the estrus cycle. The specific binding, however, is seen to decrease with the progression of pregnancy. Scatchard analysis performed with varying amounts of 125I-P-SAS in the presence of excess cold P-SAS revealed that the binding occurs with a Ka = 1.69 × 108 M-1. As P-SAS binds specifically to sialic acids on the stromal cell surface, further characterization of the sialic acid molecule to which P-SAS binds was carried out by gas liquid chromatography (GLC). The studies revealed that P-SAS preferentially binds to N-glycolylneuraminic acid, which is attached to the penultimate sugar of the stromal cell surface glycoprotein chain via 2,6 linkage. As P-SAS is further known to be mitogenic [2], the effect of P-SAS on cultured stromal cells was studied in vitro. The growth regulatory assays revealed that P-SAS induced 3H-thymidine uptake by stromal cells in culture. Thus, from the above observations, paracrine effects of P-SAS on the stromal cells and on the subsequent growth and development of the uterus can be assumed. 相似文献
8.
Sialate-O-acetylesterase was purified almost 900-fold from particle-free supernatants of horse liver by gel filtration, ion-exchange
chromatography and isoelectric focussing. The native enzyme on gel filtration exhibits a molecular weight of 54,000 Da. It
was separated by isoelectric focussing into two forms with pI values of 4.8 and 5.7, respectively. The esterase with a lower pI hydrolyses only 9-O-acetyl groups from sialic acids (KM 1.1 mM), while that with the higher pI esterifies both 4- and 9-O-acetylated monosaccharides at similar rates (KM 0.3 M and 1.3 mM, respectively). Both forms are inactive with 7-O-acetylated N-acetylneuraminic acid. Enzyme assays were carried out at the pH optimum (pH 8.4–8.6) using free O-acetylated sialic acids followed by direct analysis of the reaction products by isocratic anion-exchange HPLC. Glycosidically
bound sialic acids can also be de-O-acetylated. Horse liver esterase seems to be an essential enzyme for the catabolism of 4-O-acetylated sialoglycoconjugates, since sialidase from this tissue cannot act on 4-O-acetylated sialic acids. 相似文献
9.
Giorgos A Fragkiadakis Emmanoel K Stratakis 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1997,117(4):545-552
A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [35S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population. 相似文献
10.
Anthony P Corfield Clarice do Amaral Corfield Margret Wember Roland Schauer 《Glycoconjugate journal》1985,2(1):45-60
Clostridium perfringens sialidase is adsorbed by sialic acid immobilized on adipic acid dihydrazido-Sepharose 4B and/or polymethylacrylic hydrazido-Sepharose 4B, through its carboxyl group, C-7 to C-9 side chain, or its amino function asd-neuraminic acid--methyl glycoside or 2-deoxy-2,3-didehydroneuraminic acid. Sialidase binding was strongest to the amino-linked adsorbents, but purification was low and the enzyme could not be eluted with substrate or free sialic acid. Low binding of the sialidase to the non-substituted, blocked supports suggested that hydrophobic interactions were involved, and this was confirmed by adsorption of the enzyme on alkyl agaroses with approximately 80% of total sialidase adsorbed on decyl-agarose. Genuine affinity chromatography of sialidases is possible on immobilized sialyl-glycoconjugates, andC. perfringens sialidase could be purified to the same specific activity as the electrophoretically homogeneous enzyme using submandibular gland mucus glycoprotein adsorbents. Sialidases fromVibrio cholerae, Arthrobacter ureafaciens, Newcastle disease virus, Fowl plague virus and Influenza A2 virus also bound to immobilized sialic acids and sialyl-glycocojugates.Dedicated to Prof. Dr. Hans Faillard on the occasion of his 60th birthday. 相似文献
11.
Reinhard G. Kleineidam Olaf Hofmann Gerd Reuter Roland Schauer 《Glycoconjugate journal》1993,10(1):116-119
Fractionation of horse liver homogenate by centrifugation into heavy membranes at 10 000 × g, microsomal fraction at 105 000 × g, and the supernatant revealed sialate 9-O-lactoyltransferase activity only in the latter fraction. For the enzyme assay, the various fractions were incubated with14C labelled CMP-N-acetylneuraminic acid,N-acetylneuraminic acid and glycoconjugate-boundN-acetylneuraminic 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-boundN-acetylneuraminic acid was the best substrate tested, although some lactoylation was also found when using CMP-N-acetylneuraminic acid. 相似文献
12.
Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22 总被引:12,自引:0,他引:12
Sørge Kelm Roland Schauer Jean-Claude Manuguerra Hans-Jürgen Gross Paul R. Crocker 《Glycoconjugate journal》1994,11(6):576-585
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 theN-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 theN-formyl derivative, whereas aN-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.Dedicated to Professor Dr Gerhard Uhlenbruck on the occasion of his 65th birthday. 相似文献
13.
Mathew A. Maliakal Mepur H. Ravindranath Reiko F. Irie Donald L. Morton 《Glycoconjugate journal》1994,11(2):97-104
In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD3 and GD2) are not involved because their 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of 2,8 linkages byV. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca2+ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.Presented at the Second International Glycobiology Symposium which was held in San Francisco, CA, USA (14 February 1994). 相似文献
14.
Gerd Reuter Roland Schauer Reginaldo Prioli Miercio E A Pereira 《Glycoconjugate journal》1987,4(4):339-348
In the culture supernatant ofTrypanosoma rangeli, strain El Salvador, a sialidase was present with an activity of 0.1 U/mg protein as determined with the 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid as substrate. This enzyme was purified about 700-fold almost to homogeneity by gel chromatography on Sephadex G-100 and Blue Sepharose, and affinity chromatographies on 2-deoxy-2,3-didehydroneuraminic acid and horse submandibular gland mucin, both immobilized on Sepharose. The pH optimum is at 5.4–5.6, and the molecular weight was determined by gel chromatography, high performance liquid chromatography and sodium dodecyl sulphate gel electrophoresis to be 70 000. The substrate specificity of the enzyme is comparable to bacterial, viral and mammalian sialidases with cleavage rates for the following substrates in decreasing order:
N-acetylneuraminyl-(2–3)-lactose>
N-glycoloylneuraminy-(2–3)-lactose>
N-acetylneuraminyl-(2–6)-lactose >sialoglycoproteins>gangliosides>9-O-acetylated sialoglycoproteins.4-O-Acetylated derivatives are resistant towards the action of this sialidase. The enzyme activity can be inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Hg2+ ions, andp-nitrophenyloxamic acid; it is not dependent on the presence of Ca2+ Mn2+ or Mg2+ ions.Abbreviations BSA
bovine serum albumin
- BSM
bovine submandibular gland mucin
- CMP
cytidine monophosphate
- EDIA
ethylenediaminetetraacetic acid
- ESM
equine submandibular gland mucin
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- HPLC
high performance liquid chromatography
- Lac
lactose
- MU-Neu5Ac
4-methylumbelliferyl glycoside of -N-acetylneuraminic acid
- Neu5Ac
N-acetylneuraminic acid
- Neu5Ac2en
2-deoxy-2,3-didehydro-N-acetylneuraminic acid
- Neu4Ac5Gc
N-glycoloyl-4-O-acetylneuraminic acid
- Neu2en
2-deoxy-2,3-didehydroneuraminic acid
- Neu5Gc
N-glycoloylneuraminic acid
- PMSF
phenylmethylsulfonyl fluoride
- PSM
pig submandibular gland mucin
- SDS
sodium dodecyl sulfate
- Tris
tris-(hydroxymethyl)aminomethane
Dedicated to Professor Dr. Heinz Mühlpfordt on the occasion of his 65th birthday. 相似文献
15.
Panayotis A. Siskos Maria-Helen E. Spyridaki 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,724(2):346
A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C18 column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva. 相似文献
16.
Non-acid and acid glycosphingolipids were isolated from feces of one litter of germ-free rats from day 17 to day 51. Quantitative and qualitative changes described for small intestine of conventional rats [Bouhours D, Bouhours J-F (1981) Biochem Biophys Res Commun 99:1384–89] were also found in the feces of these germ-free rats. A decrease in lactosylceramide and sialyllactosylceramide excretion and a change fromN-acetylneuraminic acid toN-glycoloylneuraminic acid, as well as an appearance of type 1 chain blood group H-active penta- and decaglycosylceramides were observed during the weaning period. Thus the dramatic changes seen in rat intestinal glycosphingolipids postnatally seem to be primarily regulated by non-microbial factors.Abbreviations GM3
GM3-ganglioside, II3NeuAc-LacCer or II3NeuGc-LacCer
- SPG
IV3NeuAc-nLcOse4Cer
- GM1
GM1-ganglioside, II3NeuAc-GgOse4Cer 相似文献
17.
K99 Fimbriae from enterotoxigenicEscherichia coli (ETEC) were found to bind specifically to sialic acid, as measured in a haemagglutination inhibition assay using the intact bacteria and human erythrocytes. The affinity forN-glycolylneuraminic acid was about twice that ofN-acetylneuraminic acid (NeuAc), and other monosaccharides were found to be at least ten-fold less effective as inhibitors. The specificity was found to depend on electrostatic interaction where the carboxyl group and its orientation plays an important role. 2--Benzyl-NeuAc was a better inhibitor than 2--methyl-NeuAc suggesting a hydrophobic patch near the binding site on the protein. Axially oriented hydroxyl groups as in 4-epi-NeuAc and 3-hydroxy-NeuAc seemed to participate in binding since these derivatives were better inhibitors thanN-acetylneuraminic acid. K99 was found to have a higher affinity for 4-O-acetyl-NeuAc and lower affinity forN-acetylneuraminic acid withO-substituents at C7-C9 as compared toN-acetylneuraminic acid. Hence, the degree ofO-acetylation of sialic acid in the mucosa of the small intestine may influence colonization and determine susceptibility to infection. 相似文献
18.
The dominant glycosylation mutants of MDAY-D2 mouse lymphoma cells, designated class 2 (D33W25 and D34W25) were selected for their resistance to the toxic effects of wheat germ agglutinin (WGA) and shown to express elevated levels of Neu5Gc. In accordance with this, the activity of CMP-Neu5Ac hydroxylase was found to be substantially higher in the mutant cells. The hydroxylase in the D33W25 mutant cells exhibited kinetic properties identical to those of the same enzyme from mouse liver. Growth rate experimentsin vivo andin vitro, where the mutant cells grew more slowly at low cell densities in serum-free medium and also formed slower growing tumours in syngeneic mice, indicate that CMP-Neu5Ac hydroxylase expression may be associated with altered growth of the mutant cells.Abbreviations WGA
wheat germ agglutinin
- Neu5Ac
N-acetyl--d-neuraminic acid
- Neu5Gc
N-glycology--d-neuraminic acid
- CMP-Neu5Ac
cytidine-5-monophospho-N-acetylneuraminic acid
- CMP-Neu5Gc
cytidine-5-monophospho-N-glycoloylneuraminic acid
- FACS
fluorescence-activated cell sorting
- buffer A
triethylamine hydrogen carbonate, pH 7.6 (concentration given at appropriate points in the text)
- SFM
serum free medium
- IMDM
Iscove's modified Dulbecco's medium
- CMP-Neu5Ac hydroxylase
CMP-N-acetylneuraminate: NAD(P)H oxido-reductase (N-acetyl hydroxylating) (EC 1.14.99.18); CMP-sialate hydrolase (EC 3.1.4.40); sialic acid-pyruvate lyase (EC 4.1.3.3) 相似文献
19.
Nina V Prokazova Irina A Mikhailenko Sergey N Preobrazhensky Vadim O Ivanov Sergey N Pokrovsky Natalia G Timofeeva Maria A Martinova Vadim S Repin Lev D Bergelson 《Glycoconjugate journal》1986,3(3):273-286
The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous GM1- and GD1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (GM3, GD1a, GT1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (GM1, GM2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH
mouse ascites hepatoma 22a
- LDL
low density lipoprotein
- ASM
anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine]
- RITC
rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: GM3, II3NeuAc-LacCer, II3-N-acetylneuraminosyllactosylceramide
- GM2
II3-NeuAc-GgOse3Cer, II3-N-acetylneuraminosylgangliotriaosylceramide
- GM1
II3-NeuAc-GgOse4Cer, II3-N-acetylneuraminosylgangliotetraosylceramide
- GD1a, II3
IV3(NeuAc)2-GgOse4Cer, II3, IV3-di(N-acetylneuraminosyl)gangliotetraosylceramide
- GT1b
II3(NeuAc)2, IV3 NeuAc-GgOse4Cer, II3-di-N-acetylneuraminosyl, IV3-N-acetylneuraminosylgangliotetraosylceramide 相似文献
20.
A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of125I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of125I-LFA is not inhibited by 106-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 l of serum were sufficient for the detection of major gangliosides.Abbreviations LFA
Limax flavus agglutinin
- ELLA
Enzyme Linked Lectin Assay
- PIM
Poly(isobutyl methacrylate)
- PVP
Polyvinylpyrrolidone mol.wt. 40,000
- PBS
Phosphate buffered saline
- BSA
Bovine serum albumin 相似文献