Glycolipid-lectin interactions: reactivity of lectins from Helix pomatia, Wisteria floribunda, and Dolichos biflorus with glycolipids containing N-acetylgalactosamine

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.     

A new solvent system for the separation of neutral glycosphingolipids

A solvent system and a column for high performance liquid chromatography for the separation of glycosphingolipids without derivatization is described. A column pakced with porous silica gel (latrobeads) and eluted with a mixture of isopropanol-hexane-water with increasing water content and decreasing hexane content was used. Glycosphingolipids with mono- to dodeca- or tetrakaidecasaccharides were separated within 60 min and the separation pattern was highly reproducible. The method was applied for preparative separation of highly complex glycolipids with blood group activity.     

One-step purification of murine IgM and human alpha 2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.     

Purification of the beta subunit of the fibronectin receptor

In this work we describe a method for purification of the beta subunit of the mouse fibronectin receptor (GP135). Cellular glycoproteins were isolated from a detergent extract of SR-Balb tumor cell membranes by two steps of affinity chromatography on lentil lectin-Sepharose and wheat-germ-agglutinin--agarose. This material was subsequently bound to an Affi gel 102 column and eluted by increasing salt concentration. Most of the GP135 was eluted at 80 mM sodium chloride together with a few other components. A final step of hydroxyapatite chromatography in sodium dodecyl sulphate allowed elution of GP135 as a single chromatographic peak. Fractions containing GP135 were identified at each chromatographic step by immunoblotting with a specific antiserum. By this procedure GP135 was purified to homogeneity as judged by SDS-PAGE analysis of 125I-labelled material.     

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.     

Isolation of an arabinogalactan protein by lectin affinity chromatography on tridacnin-sepharose 4B.

An arabinogalactan protein from the style canal of Gladiolus has been isolated by a one-step procedure involving lectin affinity chromatography, using the galactose-binding lectin from the small giant clam, Tridacna maxima, coupled to Sepharose 4B. The lectin binding is calcium-ion dependent, so that the arabinogalactan protein which is bound to the column in the presence of calcium can be eluted by washing the column with a calcium-free buffer. This method has a general utility for the purification of polysaccharides and glycoproteins containing β-linked galactosyl residues.     

Contribution of glycolipids to species-specific antigens on erythrocytes of several animal species as to recognition of antigens with rabbit anti-glycolipids and anti-erythrocyte antisera

Because anti-glycolipid antibodies are involved in the onset of several neurological diseases, the reactivity of glycolipids on erythrocytes and the probability of generating the antibodies were determined to clarify the contribution of glycolipids as antigens. Anti-erythrocyte antisera reacted with the following glycolipids in a species-specific manner, i.e. blood group A-active glycolipid for man, Forssman glycolipid for sheep, Gg₃Cer for guinea pig, and Gg₄Cer and fucosyl GM1 for rat, and the hemolytic activities of the anti-erythrocyte antisera were attenuated by absorption of the antisera with liposomes prepared from the lipids of erythrocytes to the following levels, 94.5% for man, 24.5% for sheep, 17.5% for guinea pig, and 54.5% for rat. These species-specific glycolipids on erythrocytes reacted well with the respective anti-glycolipid antisera, but Gb₄Cer in man and GM1 in rat were shown to be cryptic on immunization with erythrocytes, indicating that the contribution of glycolipids as erythrocyte antigens differs among animal species. The glycolipid nomenclature is based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [1]. The ganglioside nomenclature of Svennerholm is employed throughout [2].     

Use of ethanol-eluted hydrophobic interaction chromatography in the purification of serum amyloid A.

A two-step procedure for the purification of the acute-phase reactant serum amyloid A from serum is described. A hydrophobic interaction chromatography medium, octyl-Sepharose CL4B, eluted with increasing concentrations of EtOH was used as the first step in the purification. The concentrate from this step was applied to a gel filtration column of Sephacryl S-200 and eluted with 10% formic acid. The overall recovery of purified serum amyloid A from serum was 56%. This represents the first time that serum amyloid A has been purified without the use of high concentrations of guanidine or urea. The method presented could easily be scaled up to allow the purification of large quantities of serum amyloid A or readily adapted to the purification of other serum apolipoproteins.     

Purification of tissue forms (Amastigotes) of Trypanosoma cruzi by immunoaffinity chromatography

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Sepharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

Purification and characterization of N-acetyl-D-galactosamine-binding lectin from Falcata japonica

An N-acetyl-D-galactosamine-binding lectin from Falcata japonica seeds was purified by affinity column chromatography of N-acetyl-D-galactosamine coupled to epoxy-activated Sepharose 6B. A 1000-fold purification of lectin was obtained from the crude extracts. The purified lectin agglutinated blood group A red cells, but neither blood group B nor O red cells. Polyacrylamide gel electrophoresis of the lectin showed one diffuse band. Molecular weights of 125,000 and 117,000 were estimated by gel filtration and ultracentrifugal analysis, respectively. SDS-polyacrylamide gel electrophoresis of the lectin also showed a single band which has a molecular weight of 34,000. Therefore, the lectin molecule was estimated to be a tetramer composed of four identical non-covalently bound subunits. F. japonica lectin was a glycoprotein containing 5% total carbohydrate, and the amino acid composition was characterized by a high content of aspartic acid, serine and glycine, a low content of methionine and the absence of cysteine.     

An improved method for tissue long-chain acyl-CoA extraction and analysis

We report an extensively modified method for the extraction, solid-phase purification, and HPLC analysis of long-chain acyl-CoAs from tissues. Tissue samples were homogenized in a glass homogenizer in KH2PO4 buffer (100 mM, pH 4.9) and again after the addition of 2-propanol. Acyl-CoAs were then extracted from the homogenate with acetonitrile (ACN). The acyl-CoAs in the extract were bound to an oligonucleotide purification column and eluted using 2-propanol. This eluent was concentrated and then loaded onto a C-18 column and eluted using a binary gradient system in which solvent A was KH2PO4 (75 mM, pH 4.9) and solvent B was ACN containing 600 mM glacial acetic acid. Initial flow rate was 0.5 or 0.25 ml/min depending upon the tissue used. The HPLC eluent was monitoring at 260 nm. Our modifications increased the recovery of the extraction procedure to 70-80%, depending upon tissue, with high reproducibility and significantly improved separation of the most common unsaturated and saturated acyl-CoAs. We also report, for the first time, the mass (nanomoles per gram wet weight) of the most common polyunsaturated acyl-CoAs in rat heart, kidney, and muscle tissues. The modifications and high recovery permit the use of tissue samples of less than 100 mg, making this method useful for the analysis of small tissue amounts associated with mice.     

Branched blood group A-active fucolipids of hog gastric mucosa.

New complex glycolipids have been extracted from hog gastric mucosa with the mixture of 0.4 M sodium acetate in methanol/chloroform/water. Three A-active fucolipids having branched carbohydrate chains have been purified from this extract. The postulated structures of these glycolipids are based on the results of partial acid hydrolysis, oxidation with periodate and chromium trioxide, and permethylation studies.     

Purification and properties of blood group A-active glycoprotein from oyster viscera

A blood group A active substance was isolated from an acetone-dried powder of oyster viscera by extraction with 0.1 M NaCl after heating a homogenate with extraction medium, in boiling water. After the removal of the acidic fraction with cetylpyridinium chloride, the separated neutral fraction was digested successively with α-amylase and amyloglucosidase to remove glycogen. The blood group A-active portion was eluted from a Sepharosé 4B column and purified by DEAE-Sephadex column chromatography. The purified active substance was homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated as 100 000 by sedimentation equilibrium.The sugar content of the purified active substance, expressed in percentage of dry weight, was galactosamine, 16.6; galactose, 12.8; fucose, 9.9; glucosamine, 4.6; and glucose, 3.3. Sialic acid was not detected. Total amino acid content was 23.0% and the main constituents were threonine, proline and serine. The ORD spectrum indicated that the hexosamines were N-acetylated. Absence of glycolipid was confirmed by the analysis of fatty acid and sphingosine base.This active substance had a strong blood group A activity (0.04 μg/ml) but neither B nor H activity; it interacted with lima bean lectin but not with concanavalian. A.     

Rapid and sensitive high-performance liquid chromatographic method for busulfan assay in plasma

A reversed-phase liquid chromatographic method with ultraviolet detection has been developed to determine busulfan concentrations in plasma of children undergoing bone marrow transplantation. Plasma samples (200 μl) containing busulfan and 1,6-bis(methanesulfonyloxy)hexane as an internal standard were prepared by a simple derivatization method with diethyldithiocarbamate followed by extraction with ethyl acetate and solid-phase purification on C₈ columns conditioned with methanol and water and eluted with acetonitrile (recovery 99%). Chromatography was accomplished using a Hypersil octadecylsilyl column (10 cm×4.6 mm I.D.) and a mobile phase of acetonitrile, tetrahydrofuran and distilled water (65:5:30, v/v). The limit of detection was 25 ng/ml (signal-to-noise ratio of 5). Calibration curves were linear up to 25 000 ng/ml. Intra-day and inter-day coefficients of variation of the assay were ≤5%. This method was used to analyse busulfan plasma concentrations after oral administration within the framework of therapeutic drug monitoring and pharmacokinetic studies in children.     

Binding of human liver hydrolases by immobilized lectins.

The binding of 22 human liver hydrolase activities by immobilized lectins of six different carbohydrate specificities, namely alpha-D-mannose (glucose), D-N-acetylglucosamine, D-N-acetylgalactosamine, L-fucose, alpha-D-galactose and beta-D-galactose, were examined. Differences in binding among these enzymes and within specific enzymes were observed. For example, the neutral forms of alpha-mannosidase and beta-xylosidase were bound by the Ulex europaeus lectin I (specific for L-fucose), whereas the acidic forms were not. Bandierea simplicifolia lectin (specific for alpha-galactose) bound 65% of beta-glucuronidase activity; recycling experiments demonstrated complete binding of the enzyme that had been eluted with the competitor D-galactose and no binding of the fraction that was not initially bound. These results suggested the presence of two forms of this enzyme. Similar data were obtained for acidic beta-galactosidase activity. These experiments may provide the basis for the expanded use of immobilized lectins for purification and characterization of hydrolases and other glycoproteins.     

Separation and characterization of the acid lipase and neutral esterases from human liver.

Electrophoresis of human liver homogenates followed by reaction with 4-methylumbelliferyl palmitate reveals the presence of two major electrophoretic forms with esterase (lipase) activity toward this substrate. The two enzymes were isolated and partially purified based on their solubility differences and their relative affinities for the lectin column concanavalin A-Sepharose 4B. Lipase A was particulate with an acidic pH optimum (5.2) and could be solubilized with the non-ionic surfactant Triton X-100. Lipase B was soluble and had a more neutral pH optimum (6.3--6.6). Both forms bound to immobilized concanavalin A and could be specifically eluted. Buffers containing alpha-methylmannoside eluted lipase B, and buffers with alpha-methylmannoside and Triton X-100 eluted lipase A, giving a 22- and 257-fold purification, respectively, over whole-tissue homogenates. Cholesterol oleate, trioleoylglycerol, and 4-methylumbelliferyl palmitate were substrates for solubilized lipase A. Lipase B hydrolyzed 4-methylum-belliferyl palmitate but not trioleoylglycerol or cholesterol oleate. Lipase B was more thermolabile than lipase A, and it was selectively inhibited by diethyl-p-nitrophenyl phosphate at low concentrations. We conclude that lipase A and B are distinctly different enzymes and that they are probably not related polymorphic forms of one another.     

One-step fractionation of neutral and acidic glycosphingolipids by high-performance liquid chromatography

A procedure for a simultaneous separation of ganglioside components and neutral glycolipid components by high-performance liquid chromatography was described. One column packed with DEAE-derivatized controlled-pore glass (DEAE-CPG) was serially connected to two columns of underivatized, controlled-pore glass (CPG). A mixture of gangliosides and neutral glycolipids were loaded on DEAE-CPG and eluted with a mixture of chloroform-methanol-water, with increasing methanol and water (the first-phase gradient elution), followed by elution with increasing concentrations lithium acetate from 0.015 to 0.1 M in a mixture of chloroform-methanol-water (the second-phase gradient elution). Neutral glycolipids, mono- to hexaglycosylceramides , were separated within 80 min of the first-phase gradient elution, and mono- to tetrasialosylgangliosides were separated during the second-phase gradient elution within 60 min. The method has been applied to the determination of glycolipids isolated from rat tissues, and the procedure was found to be highly reproducible.     

Liquid chromatographic analysis and preliminary pharmacokinetics of methotrexate in cancer patients co-treated with docetaxel

A new HPLC method has been developed for the quantitative determination of methotrexate (MTX) and its 7-hydroxyl metabolite in human plasma. Samples were purified by protein precipitation with acetone and methanol, and a sample clean-up with a mixture of n-butanol and diethyl ether. The analytes were separated on an RP Inertsil ODS-80A column and eluted in a solvent system containing 5% (v/v) tetrahydrofuran in water (pH 2.0). UV absorption measurement was performed at 313 nm, and the detector response was linear in a concentration range of 10–10 000 ng/ml. The lower limit of quantitation of MTX was 10 ng/ml using 1 ml sample aliquots. Values for accuracy and (within-run and between-run) precision were between 95.5–111% and 3.69–11.0%, respectively, at four concentrations analyzed in quintuplicate on four separate occasions. The assay was applied to study the effects of docetaxel co-administration on the pharmacokinetics and metabolism of MTX in cancer patients.     

Transient expression of type 2 chain in A-active hexaglycosylceramide of rat small intestine at weaning time. Demonstration by affinity chromatography and ceramide glycanase hydrolysis of A-active glycosphingolipids followed by gas chromatography and mass spectrometry of permethylated hexasaccharides

The small intestine of 15- to 23-day-old rats was cut into four segments from the duodenum to the ileum. Neutral glycosphingolipids were purified from each segment and submitted to thin-layer chromatography and immunostaining with the A005 monoclonal anti-A antibody. This antibody detected an hexaglycosylceramide located mainly in the duodenum during the postnatal development. In order to characterize hexaglycosylceramides, blood group A-active glycolipids were purified by affinity chromatography on immobilized Helix pomatia lectin in organic solvent. Hexaglycosylceramides (A-6) were subsequently isolated by preparative thin-layer chromatography and hydrolyzed with ceramide glycanase. The free hexasaccharides were permethylated and analyzed by gas chromatography. Two peaks were detected in varying ratios during development, corresponding to type 1 and type 2 chain A hexasaccharides. Gas chromatography clearly demonstrated that type 2 A-6 occurred in the duodenum of developing rats, and that a shift from type 2 to type 1 A-6 occurred with growing age. The change from type 2 to type 1 chain was also assessed by methylation analysis, and by the variation of the characteristic fragmentations of type 1 and type 2 chain hexasaccharides upon mass spectometry of the permethylated A-6 oligosaccharides from the duodenum of 19-day-old and adult rats.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.