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1.
The autoradiographic detection of 125I-labeled lectins binding to glycolipids on thin-layer chromatograms can be used to rapidly analyze total glycolipid extracts of cells or tissues for specific oligosaccharide structures. The Helix pomatia lectin which binds with high affinity to terminal alpha-linked GalNAc residues did not bind to globoside (terminal beta 1-3GalNAc) but did bind the ganglioside GM2 and its asialo derivative which have terminal beta 1-4GalNAc residues. The lectin from Dolichos biflorus bound specifically to the Forssman glycolipid with relatively low affinity. The lectin from Wisteria floribunda was bound to Forssman glycolipid, globoside, and the asialo derivative of the ganglioside GM2. The interactions of these lectins with the glycolipid-derived, 3H-labeled oligosaccharides was also analyzed by affinity chromatography. The results indicated that the reactivity of multivalent carbohydrate-binding proteins with polyvalent surfaces of glycolipids is strong enough to permit detection of low-affinity interactions that may not be observed in binding assays that are based on carbohydrate-protein interactions in solution. The autoradiographic analysis of 125I-Helix pomatia lectin binding to thin-layer chromatograms of total lipid extracts from human erythrocyte membranes detected the quantitative differences in the A-active glycolipids from type A1 and A2 cells. 相似文献
2.
Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates 总被引:5,自引:0,他引:5
Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes. 相似文献
3.
Glycolipid antigens of human polymorphonuclear neutrophils and the inducible HL-60 myeloid leukemia line 总被引:10,自引:0,他引:10
F W Symington D L Hedges S Hakomori 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(4):2498-2506
Glycolipid and cell surface carbohydrate antigens of human polymorphonuclear neutrophils (PMN) and of HL-60 myeloid leukemia cells were analyzed with a panel of defined, monoclonal anti-carbohydrate antibodies. Antigenicities of intact PMN, HL-60, and retinoic acid-induced HL-60 (r.a.-HL-60) were studied by flow cytofluorometry. These three cell populations displayed quantitative differences, some of which were induction dependent, in their expression of lactosyl, N-acetyllactosaminyl, Y-hapten (Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R), and sialosyl-X-hapten (SA alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R) specificities. Structures reactive with antibodies specific for long-chain mono-, and di- or tri- alpha 1----3 fucosylated lacto-series glycolipids were also detected. Glycosphingolipids purified from organic extracts of these cells were analyzed to seek information concerning the chemical basis for these surface antigenic differences, to assess the structural and antigenic diversity of PMN and HL-60 glycolipids, and to quantitate chemically and antigenically prominent glycolipids. Binding of monoclonal antibodies to thin-layer chromatograms demonstrated that each of the specificities on intact cells was carried by one or more distinct glycolipids. The abundance of immunoreactive glycolipids in the extracts paralleled the relative staining intensities of the intact cell populations. Several "cryptic" glycolipid antigens, including alpha 2----6 sialosylated structures enriched five- to 10-fold in PMN extracts, were not detected on intact cells. Lactosylceramide accounted for two-thirds of the approximately 1.5 X 10(9) glycolipid molecules contained in each PMN. The remaining glycolipid antigens appeared to include structurally diverse fucolipids, fucogangliosides, and neutral and sialosylated glycolipids with Gal beta 1----4GlcNAc beta 1----R terminal core structure. The abundance, diversity, and induction-dependent expression of these structures suggest that they may participate in PMN maturation and function. 相似文献
4.
A novel tumor-associated, developmentally regulated glycolipid antigen defined by monoclonal antibody ACFH-18 总被引:1,自引:0,他引:1
E D Nudelman S B Levery M R Stroud M E Salyan K Abe S Hakomori 《The Journal of biological chemistry》1988,263(27):13942-13951
A mouse IgM monoclonal antibody, ACFH-18, was established after immunization of mice with the human gastric cancer cell line MKN74. The antibody reacts strongly with gastrointestinal carcinoma and showed a clear dependence on the degree of differentiation of gastric cancer cells. The antibody defines a series of glycolipid species with extremely slow TLC mobility present in both acidic and neutral glycolipid fractions of the extract from gastrointestinal adenocarcinoma and the original MKN74 cells. Isolation and structural study of the active glycolipids present in the acidic and neutral fractions and comparison of the antibody reactivity with glycolipids having related structures revealed a novel specificity. The minimum requirement for the maximal reactivity of ACFH-18 was identified as VII3Fuc-nLc10 (Structure A below and Fig. 8 in text). Since the antibody did not react with III3Fuc-nLc6 (Structure B below), which shares the same terminal sequence as VII3Fuc-nLc10, and since it cross-reacted with VII3Fuc-nLc8 (Z1 glycolipid) and VII3Fuc,V3-Fuc,III3Fuc-nLc8 (Z3 glycolipid), antibody ACFH-18 is capable of recognizing a fucosyl residue plus an internal repeating N-acetyllactosamine proximal to ceramide, as indicated by lines in Fig. 8 (Structures A, B, and E-G). (Formula: see text). 相似文献
5.
The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species. 相似文献
6.
M N Fukuda A Dell J E Oates P Wu J C Klock M Fukuda 《The Journal of biological chemistry》1985,260(2):1067-1082
Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes. 相似文献
7.
A series of human erythrocyte glycosphingolipids reacting to the monoclonal antibody directed to a developmentally regulated antigen SSEA-1 总被引:23,自引:0,他引:23
R Kannagi E Nudelman S B Levery S Hakomori 《The Journal of biological chemistry》1982,257(24):14865-14874
A series of glycolipid antigens reacting with the monoclonal antibody directed to the stage-specific embryonic antigen 1 was isolated and characterized from group O human erythrocyte membranes. A ceramide heptasaccharide (Structure 1), ceramide nonasaccharide (Structure 2), and ceramide decasaccharide (Structure 3) have been characterized (formula, see text) The main feature of this glycolipid series is its long core sugar chain with a nonbranched repeating N-acetyllactosamine (norpolylactosamine). This characteristic is in contrast to that of co-existing H-active glycolipid series in which the longer core structures are branched type repeating N-acetyllactosamine (isopolylactosamine). The reactivity of these glycolipids to monoclonal anti-stage-specific embryonic antigen 1 antibody varied proportionately to the length of their core sugar chains. A possible significance of these glycolipids as developmentally regulated antigens and as cancer-associated antigens was discussed. 相似文献
8.
Mo H Rice KG Evers DL Winter HC Peumans WJ Van Damme EJ Goldstein IJ 《The Journal of biological chemistry》1999,274(47):33300-33305
An unusual lectin possessing two distinctly different types of carbohydrate-combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e. asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that the X. sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (alpha(2)beta(2)) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal alpha1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide with N-acetyllactosamine (i.e. Galbeta1,4GlcNAc-) or lacto-N-biose (i.e. Galbeta1,3GlcNAc-) groups at its three nonreducing termini. 相似文献
9.
Isolation and characterization of a family of alpha-D-galactosyl-containing glycopeptides from Ehrlich ascites tumor cells 总被引:8,自引:0,他引:8
A family of glycopeptides that contain nonreducing terminal alpha-D-galactosyl residues has been isolated from Pronase digests of delipidated Ehrlich ascites tumor cells. The glycopeptides, which comprise 17.2% of the total plasma membrane hexose, have an average molecular weight of 7500 and are precipitated by Griffonia simplicifolia B4 isolectin, wheat germ agglutinin, and Ricinus communis lectin. Exo- and endoglycosidase digestion, periodate oxidation, permethylation analysis, and lectin reactivity provided evidence for a tentative carbohydrate structure for the glycopeptide mixture. The glycopeptides possess tetraantennary branched structures containing a trimannosyl core N-glycosidically linked via an N,N'-diacetylchitobiosyl unit to an asparagine residue. Each branch contained repeating leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units resulting in a keratan-like structure, terminated with alpha-D-Galp-(1 leads to 3)-[alpha-D-Galp-(1 leads to 6)]-beta-D-Galp-units. The variation in the molecular weight observed for the glycopeptide mixture can be attributed to the variable amounts of leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units found in the branch chains. 相似文献
10.
Human serum was shown to contain N-acetyllactosamine: N-acetylglucosaminyltransferase activity. The reaction product was hydrolyzed by beta-N-acetylglucosaminidase and released [14C]N-acetylglucosamine, indicating that the N-acetylglucosaminyl residue was beta-linked to N-acetyllactosamine. Methylation and hydrolysis of the reaction product yielded 2,4,6-trimethyl[3H]galactose, indicating that the N-acetylglucosaminyl residue was introduced at position C-3 of the terminal galactose of N-acetyllactosamine. In our experiments, 2,3,4-trimethyl[3H]galactose was not detected. Substrate competition studies between N-acetyllactosamine and lactose showed that this enzyme also catalyzed the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to lactose. Since the Km value for N-acetyllactosamine, which was 7.0 mM, was approximately a fourth of that for lactose (29.8 mM), N-acetyllactosamine was more effective than lactose as an acceptor. 相似文献
11.
Campylobacter pylori colonization factor shows specificity for lactosylceramide sulfate and GM3 ganglioside 总被引:8,自引:0,他引:8
B L Slomiany J Piotrowski A Samanta K VanHorn V L Murty A Slomiany 《Biochemistry international》1989,19(4):929-936
The specificity of Campylobacter pylori cell surface lectin, a presumptive colonization factor, was investigated using various sulfated and sialic acid containing glycolipids. C. pylori cells, cultured from human antral mucosal biopsies, were incubated with intact and modified glycolipid preparations and examined for agglutination inhibition of human erythrocytes. Titration data revealed that the inhibitory activity was highest with lactosylceramide sulfate and GM3 ganglioside, while galactosylceramide sulfate GM1, GD1a and GD1b gangliosides were less effective. A strong inhibitory activity towards C. pylori hemagglutin was also observed with an antiulcer agent, sucralfate. The inhibitory effect of both types of glycolipids was abolished by the removal of sialic acid and sulfate ester groups, thus indicating that sulfated and sialic acid containing glycolipids with terminal lactosyl moieties serve as mucosal receptors for colonization of gastric epithelium by C. pylori. 相似文献
12.
The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans. 相似文献
13.
Human erythrocyte polyglycosylceramides (PGCs) are recognized by the gastric pathogen Helicobacter pylori and are based on a successively extended and highly branched N-acetyllactosamine core linked to ceramide and substituted by fucose and sialic acid. As a step in the identification of the binding epitope we earlier characterized intact PGCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS (Karlsson,H., Johansson,L., Miller-Podraza,H., and Karlsson,K-A. [1999] Glycobiology, 9, 765-778). In the present work, PGCs from human blood group O erythrocytes were digested with endo-ss-galactosidase (Bacterioides fragilis), an enzyme which cleaves the bond 3Galss1-4GlcNAc in linear but not branched poly-N-acetyllactosamine chains. The enzymatic digestion resulted in a mixture of neutral and sialic acid-containing glycolipids together with terminal and internal sequences of mainly neutral oligosaccharides. The products were analyzed by MALDI-TOF MS in both positive and negative ion mode which gave spectra where the ions could be assigned to structures of the neutral and acidic components, respectively. Among glycolipids found were [structure in text] where R could be H, Fuc or NeuAc. Also observed were structures as [structure in text] which indicated linear extension along both branches. Observed at higher masses were fully branched structures obtained by stepwise extension with [structure in text] where R could be H, Fuc or NeuAc. Most probably further branching may occur along both the (1-->3)- and the (1-->6)-linked branches to give a partly dendritic structure. Structures with more than one sialic acid substituted could not be observed in the MALDI spectrum. Complementary information of the terminal sequences was obtained by FAB-MS analysis of permethylated undegraded PGCs. High-temperature gas chromatography/mass spectrometry of reduced and permethylated products from enzyme hydrolysis documented that Fuc was present in a blood group O sequence, Fuc-Hex-HexN-. Fucose may be placed on short (monolactosamine) or longer branches, while sialic acid seems to be restricted to monolactosamine branches. The conclusion is that human erythrocyte PGCs display microheterogeneity within terminal and internal parts of the poly-N-acetyllactosamine chains. The first branch from the ceramide end may be located at the second or third Gal and possibly also on the first Gal. Other branches may occur on every N-acetyllactosamine unit in fully branched domains, or there may be linear extensions between branches resulting in incompletely branched structures. The extended linear sequences may be present in both 3- and 6-linked antennae. Terminal structures are based on one, two or maybe higher number of N-acetyllactosamine units. 相似文献
14.
Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides. 相似文献
15.
In the lysosome, glycosidases degrade glycolipids, glycoproteins, and oligosaccharides. Mutations in glycosidases cause disorders characterized by the deposition of undegraded carbohydrates. Schindler and Fabry diseases are caused by the incomplete degradation of carbohydrates with terminal alpha-N-acetylgalactosamine and alpha-galactose, respectively. Here we present the X-ray structure of alpha-N-acetylgalactosaminidase (alpha-NAGAL), the glycosidase that removes alpha-N-acetylgalactosamine, and the structure with bound ligand. The active site residues of alpha-NAGAL are conserved in the closely related enzyme a-galactosidase A (alpha-GAL). The structure demonstrates the catalytic mechanisms of both enzymes and reveals the structural basis of mutations causing Schindler and Fabry diseases. As alpha-NAGAL and alpha-GAL produce type O "universal donor" blood from type A and type B blood, the alpha-NAGAL structure will aid in the engineering of improved enzymes for blood conversion. 相似文献
16.
Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins. Its structure and reactivity toward plant lectins 总被引:1,自引:0,他引:1
H Kitagaki-Ogawa I Matsumoto N Seno N Takahashi S Endo Y Arata 《European journal of biochemistry》1986,161(3):779-785
Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatography on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods. The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xyl beta 1----2 (Man alpha 1----6)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc by high-performance liquid chromatography, sugar analysis and 1H-NMR spectroscopy. The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish-peroxidase-conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: a beta-xylosyl residue substituted at C-2 of the beta-mannosyl residue of N-linked oligosaccharide does not affect the binding with mannose-specific lectins, lentil, pea and Vicia faba lectins can bind to N-linked oligosaccharides containing an alpha-L-fucosyl residue attached to C-3 of the asparagine-linked N-acetyl-D-glucosamine residue, and Ulex europeus agglutinin I can bind to the (alpha 1----3)-linked fucose residue of the N-linked oligosaccharide. 相似文献
17.
The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine. 相似文献
18.
Cholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a β-galactoside the liposomes are aggregated by ricin and when it is an α-mannoside they are aggregated by Con A. The lectin-mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin-mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on (1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl-containing glycolipid is independent of glycolipid concentration, and (2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane-bound receptors in order for aggregation to occur is discussed. 相似文献
19.
As a consequence of detecting an IgM M-protein (naturally occurring diseased-state monoclonal antibody) immunoreactive to insect acidic glycolipids in a patient with demyelinating peripheral neuropathy, normal human sera were examined for the occurrence of heterophile antibodies directed against carbohydrate epitopes present on glycosphingolipids of Calliphora vicina (Insecta: Diptera). The insect glycolipids can be separated into neutral, zwitterionic, and acidic types, according to whether the oligosaccharide chains consist of neutral monosaccharides only, or carry an additional phospho-ethanolamine side chain and/or a beta-glucuronic acid residue, respectively. Natural antibody activity to these three classes of insect glycosphingolipids was detected in all normal human sera examined. The antibody activities were separated by sequential chromatography on affinity columns of octyl-Sepharose 4B-bound neutral and zwitterionic glycolipids into three populations with differing epitope-type specificities. As expected for heterophile antibodies, they are mainly of the IgM class. Population I recognized epitopes present on the three types of insect glycolipids, i.e., the neutral oligosaccharide chain backbone, the main determinant of which contains a terminal N-acetylhexosamine. Immunoreactivity is separable into at least four subpopulations of differing carbohydrate epitope specificity. Population II recognized epitopes containing phosphoethanolamine in zwitterionic and some acidic insect glycolipids. There are two subpopulations, the majority of which require the free amino group of phosphoethanolamine for immunoreactivity. Population III antibodies showed immunoreactivity to terminal beta-glucuronic acid-containing epitopes present only on acidic insect glycolipids. 相似文献
20.
R N Knibbs I J Goldstein R M Ratcliffe N Shibuya 《The Journal of biological chemistry》1991,266(1):83-88
The sialic acid-specific leukoagglutinating lectin from the seeds of Maackia amurensis (MAL) has been studied by the techniques of quantitative precipitin formation, hapten inhibition of precipitation, hapten inhibition using an enzyme-linked immunosorbent assay, and lectin affinity chromatography. The ability of the immobilized lectin to fractionate oligosaccharides based on their content of sialic acid has also been investigated. Our results indicate that MAL reacts with greatest affinity with the trisaccharide sequence Neu5Ac/Gc alpha 2,3Gal beta 1,4GlcNAc/Glc. The lectin requires three intact sugar units for binding and does not interact when the beta 1,4-linkage is replaced by a beta 1,3-linkage nor when the "reducing sugar" of the trisaccharide is reduced. Results from enzyme-linked immunosorbent assays show that an N-acetyllactosamine repeating sequence is not required; however, the N-acetyllactosamine repeating sequence does appear to enhance the binding of MAL to a series of glycolipids. In addition, the sialic acid may be substituted with either N-acetyl or N-glycolyl groups without reduction in binding. The C-8 and C-9 hydroxyl groups of sialic acid do not play a role in binding as shown by the strong reaction of periodate-treated glycoproteins. Comparison of the specificity of the three sialic acid-binding lectins indicates that Limax flavus agglutinin binds to Neu5Ac in any linkage and in any position in a glycoconjugate, Sambucus nigra lectin requires a disaccharide of the structure Neu5Ac alpha 2,6Gal/GalNAc, and MAL has a binding site complimentary to the trisaccharide Neu5Ac alpha 2,3Gal beta 1,4GlcNAc/Glc, to which sialic acid contributes less to the total binding affinity than for either S. nigra lectin or L. flavus agglutinin. 相似文献