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[Study of the nature of A1- and A2-antigenic differences with use of monoclonal antibodies: a role of glycoprotein and glycolipid epitopes in their formation].

The author used of A1- and A2-red blood cells to absorb Workshop-IV monoclonal reagents (anti-A1 2-24, 2-25, 2-27; anti-A 2-4, 2-10, 2-28; anti-H 2-71, 2-74) and anti-AHP protection, inhibited them with protein glycoconjugates (Apr), obtained by treating red blood cells with trypsin at pH 6.6 and by separating the supernatant on anion-exchange columns, and with lipid glycoconjugates (Alp), obtained by isolating glycosphingolipids from the trypsin-pretreated red blood cell membranes by the chloroform-methanol method and by subsequently performing anion-exchange gel chromatography. Judging from the results of the absorption using A1- and A2-red blood cells, the antigenic spectrum of A1 is more complete and exceeds that of A2, which permitted the use of the uniform test system of glycoconjugates from A1-red blood cells. Unlike monoclonal antibody's anti-A, the anti-A1-reagents were inhibited more slightly by glycolipid conjugates (other than those of the alkaline type (A1p-0), elution area pH 8.0-8.2), but more strongly by glycoprotein conjugates of the alkaline and moderately acid types (Apr-) and Apr-1 elution area pH 7.9 and 7.1) and they also reacted with the acid type of glycoprotein and glycolipid conjugates (Apr-3 and A1p-3 elution area pH 6.5-6.7). The use trypsin-treated A1-test-red blood cells dramatically decreased the titer of anti-A1-antibodes and failed to reveal the inhibitory activity of Apr-glycoconjugates. The difference in the inhibition of antibodies by glycoconjugates of various types, as well as the selectivity in the responsiveness of anti-A1-antibodies are attributable to the fact that A2-red blood cells show attenuation of not only glycosphingolipid epitopes of the alkaline types (A1p-0), but also glycoprotein epitopes of the acid type (Apr-3) in particular.

ABO Blood-Group System↗

Glycolipids of the fish testis.

Glycolipids were purified from the total lipid extract of the testis or milt of a kind of puffer (Fugu rubripes rubripes) by adsorption column chromatography using silicic acid and magnesium silicate and by preparative silica gel TLC. The glycolipids were identified as glucosylceramide (116 mug/g wet tissue) and galactosylceramide 26.7 mug/g). Seminolipid, a sulfagalactolipid specific to mammalian testis was not detected, but the presence of a small amount of sulfatide (15.2 mug/g) was demonstrated. The long-chain bases of both cerebrosides were mainly C18-sphingenine, but in sulfatide, C20-sphingenine was more abundant than C18-sphingenine. In both cerebrosides and sulfatide, the fatty acid compositions were similar, with nervonic acid as the predominant component. Two species of gangliosides were also obtained and were identified as N-acetylgalactosaminyl(1 leads to 4)[N-acetylneuraminyl(2 leads to 3)]galactosyl(1leads to 4)glucosylceramide (59.8 mug/g) and N-acetylneuraminyl(2 leads to 3)galactosyl(1 leads to 4)N-acetylglucosaminyl(1 leads to 3)galactosyl(1 leads to 4)glucosylceramide (45.0 mug/g). The long-chain bases of the two gangliosides consisted of C18-spingenine and C20-sphingenine, and the major fatty acids were palmitic and stearic acids.

Animals↗

Association of the glycolipid pattern with antigenic alterations in mouse fibroblasts transformed by murine sarcoma virus.

The level of the neutral glycolipid, Galnac beta 1 leads to 4 Gal beta 1 leads to 4Glc leads to Cer (asialo GM2), in BALB/c 3T3 mouse fibroblasts transformed by Kirsten murine sarcoma virus (3T3KIMSV) was greatly increased compared to the nontransformed parental cells (3T3). This elevated chemical quantity was found to be localized on the surface of intact cells and accessible to external reagents, as detected by immunofluorescence and labeling with galactose oxidase: NaB3H4. Furthermore, immunization of rabbits with 3T3KiMSV cells but not with 3T3 cells resulted in antibody production against asialo GM2. These results demonstrate the potential usefulness of glycolipids as tumor-associated cell surface markers.

Antibodies, Neoplasm↗

Biochemical analysis of glycophospholipids from wild-type mouse lymphoma cells and from Thy-1 negative mutants which do not add glycolipid-anchored proteins.

Thy-1 is a major glycophospholipid (GPL)-anchored protein found on the surface of neurons, epithelial cells, fibroblasts and murine T-lymphomas. Biochemical studies were undertaken to determine if murine T-lymphomas contain glycolipids which may be on the path of GPL-anchor biosynthesis. Biosynthetic labeling experiments on Thy-1-positive (wild-type) cells followed by battery of chemical and enzymatic diagnostics on the isolated [3H]mannolipids have, for the first time, led to description of a set of glycolipids which have properties consistent with their being GPL-anchor precursors. Using these results as a guide, major differences have been observed upon analysis of the radiolabeled mannolipids of Thy-1-negative mutants from 7 complementation classes, A-C, E, F, H and I. The biosynthetic lesions in anchor synthesis have been identified in some of the mutants.

Animals↗

Structures of the glycoinositolphospholipids from Leishmania major. A family of novel galactofuranose-containing glycolipids.

Structures of the major glycolipids isolated from the protozoan parasite Leishmania major (strains V121 and LRC-L119), were elucidated by fast atom bombardment-mass spectrometry, two-dimensional proton NMR, methylation analysis, exoglycosidase digestions and mild acid hydrolysis. These glycolipids belong to a family of glycoinositolphospholipids (GIPLs), which contain 4-6 saccharide residues linked to alkylacylphosphatidylinositol (alkylacyl-PI) or lyso alkyl-PI. The general structure of the elucidated GIPLs can be expressed as follows: R-3Galf(alpha 1-3)Manp(alpha 1-3)Manp(alpha 1-4)GlcNp(alpha 1-6) alkylacyl-PI or lyso alkyl-PI where R = OH for GIPL-1; R = Galp(alpha 1- for GIPL-2; R = Galp(alpha 1-6)Galp (alpha 1- for GIPL-3 and R = Galp(alpha 1-3)Galf(alpha 1- for GIPL-A. The alkylacyl-PI lipid moieties are unusual in containing predominantly 18:0, 22:0, 24:0, or 26:0 alkyl chains and 12:0, 14:0, or 16:0 acyl chains. Remodeling of the lipid moieties may occur based on the finding that 1) lyso derivatives account for approximately 35% of the GIPL-3 fraction in strain V121 and 2) there is an increase in the proportion of 24:0 and 26:0 alkyl chains with elongation of the carbohydrate chain. Together with the elucidated structures, these properties are consistent with some of the GIPLs having a role as biosynthetic precursors to the major cell surface glycoconjugate, lipophosphoglycan. In particular, the saccharide sequences of GIPL-3, lyso-GIPL-3, and the glycan core of lipophosphoglycan (Turco, S. J., Orlandi, P. A., Homans, S. W., Ferguson, M. A. J., Dwek, R. A., and Rademacher, T. W. (1989) J. Biol. Chem. 264, 6711-6715) are identical. Finally, immunostaining of thin layer chromatograms with antibodies from patients with cutaneous leishmaniasis suggests that the major GIPLs are highly immunogenic and that the elevated anti-Gal antibodies, commonly seen in leishmaniasis patients, may be directed against terminal Galp(alpha 1-3)Galf residues.

Animals↗

Aminopropyl silica gel as a solid support for preparation of glycolipid immunoadsorbent and purification of antibodies.

Aminopropyl silica gel was prepared from porous silica gel and was used as a solid support for immunoadsorbent in the purification of anti-glycolipid antibodies. For neutral glycosphingolipids, a carboxyl function was generated by oxidation of the olefinic double bond of the sphingosine moiety, whereas for gangliosides the carboxyl group of sialic acid was used to couple with aminopropyl silica gel in the presence of a carbodiimide. These compounds were used for purifying anti-glycolipid antibodies from serum of immunized rabbits. The antibodies bound to the su-strate were released by 2 M potassium thiocyanate and their immunological properties were studied. Aminopropyl silica gel may be preferred over conventional organic solid supports for the following reasons: 1) faster flow rate; 2) higher capacity; 3) easier handling; 4) more economical; and 5) lower susceptibility to microbial attack.

Antibodies↗

[The surface glycolipid antigen specific for the internal cell mass of the mouse blastocyst and of the stem cells of murine teratocarcinoma F9].

A new monoclonal antibody that recognizes a new antigen on the surface of mouse teratocarcinoma F9 stem cells has been described. This antigen is a glycolipid as demonstrated by inhibition of immunofluorescence by different monosaccharides, glycoproteins and glycolipid fraction of F9 cells as well as by chemical analysis. Immunofluorescent staining of in vitro cultivated preimplantation mouse embryos has demonstrated that this antigen is specific only of internal cell mass cells of late blastocyst.

Animals↗

Structure and function of mycobacterial glycolipids and glycopeptidolipids.

Earlier work from this and other laboratories has revealed the presence within Mycobacterium spp. of three classes of glycolipid antigens which we have called the glycopeptidolipids, the lipooligosaccharides and the phenolic glycolipids. Representative structures of each from different species and sub-species have been proposed. More recently, new variants of these antigens and older structures have been analyzed by Fourier transform infrared, NMR, particularly at high temperatures, and, most notably, by fast atom bombardment and Californium desorption mass spectrometry. Extraordinary novelty and diversity were revealed, particularly at the distal non-reducing end of the oligosaccharide chains, marked by the presence of new branched-chain sugars, amino sugars and sugar acids. These epitopes and monoclonal antibodies to them have been used for the critical identification of mycobacteria. In addition, the pure antigens are the basis of specific serological tests for various mycobacterioses. The resurgence of interest in "atypical" mycobacteria stems from their occurrence as opportunistic pathogens in many patients with acquired immunodeficiency syndrome, although they have long been associated with pulmonary and other organ infections. Foremost among these mycobacteria are serovars of the Mycobacterium avium-Mycobacterium intracellulare complex (the M. avium complex). The surface antigens which differentiate these serovars are glycopeptidolipids, related to "mycoside C" and, accordingly, composed of a glycosylated lipopeptide "core", fatty acyl-D-Phe-D-alloThr-D-Ala-L-acanyl-O- (3,4-di-O-methyl-alpha-L-rhamnopyranoside), to which a haptenic oligosaccharide is linked at the threonine substituent; this oligoglycosyl unit is the source of type specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Serological specificity of Mycobacterium tuberculosis glycolipids.

Six glycolipid fractions can be extracted from lipid crude extracts of Canetti type strains of M. tuberculosis. Among these fractions are two phenolglycolipids. The major component is a triglycosyl phenolphthiocerol dimycocerosate (PGL-Tb 1) and the second is a monoglycosyl diacyl phenolphthiocerol identical to mycoside B of M. bovis. Similar glycolipid compounds can also be found in wild strains of M. tuberculosis recently isolated from tuberculous patients. One of these compounds has been identified as PGL-Tb 1.

Antibodies, Bacterial↗

The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid.

P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with [3H]palmitic acid and with myo-[2-3H]inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological "cross-reacting determinant" first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with [35S]methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified [3H] palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.

Amino Acid Sequence↗

Comparison of glycolipids in various human leukemia cells.

Glycolipids from human leukemia cells were analyzed by gas-liquid chromatography, enzymatic hydrolysis and high-performance thin-layer chromatography with immunostaining by the use of mouse monoclonal antibody. Glucosylceramide, lactosylceramide, and ganglioside GM3 were found in various leukemia cases. Acute lymphoblastic leukemia cells contained little or none of the glycolipids with lacto-series structures such as neolactotetraosylceramide (nLc4Cer), NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer, or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer (IV6NeuAc-nLc4Cer), which were found in every case of various myeloid leukemias. GM3 was a major ganglioside (45-100%) in acute leukemia cells, whereas IV6NeuAc-nLc4Cer was a major one (37%) in chronic myelogenous leukemia cells.

Gangliosides↗

Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides.

The variant surface glycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysis of the intact protein and of glycopeptides. Processing has been examined by comparing glycan structures purified from an immature intracellular form (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAc2) at Asn-432 in both the immature 58-kDa and mature 59-kDa forms. In contrast, the "core" oligosaccharide of Asn-419 (Man3-GlcNAc2) appears to be nearly quantitatively processed to a complex biantennary structure [Gal-GlcNAc-Man)2-Man-GlcNAc2) during VSG maturation. The asparagine-linked structures at Asn-419, but not those at Asn-432, are resistant to endo-beta-N-acetylglucosaminidase H within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This report provides a direct demonstration of the processing of such a glycolipid anchor following its attachment to protein.

Amino Acid Sequence↗

Human monoclonal antibodies to phenolic glycolipid-I derived from patients with leprosy, and production of specific anti-idiotypes.

Human monoclonal antibodies (mAb) were produced by hybridomas derived from fusion of the GM4672 lymphoblastoid cell line and peripheral blood mononuclear cells from leprosy patients. Hybridoma supernatants were screened for immunoglobulin (Ig) secretion, binding to Mycobacterium leprae, phenolic glycolipid-I (Phen GL-I), the unique M. leprae glycolipid and single-stranded(ss)DNA by ELISA. On the basis of direct-binding ELISAs, two IgMk mAb (PR4 and TH3) were selected for characterization. PR4 and TH3 bound to M. leprae, Phen GL-I and ssDNA; PR4 also bound to M. avium and M. kansasii and TH3 to M. kansasii. Inhibition assays demonstrated that these antibodies did not bind to the terminal disaccharide of Phen GL-I. In addition, both PR4 and TH3 bound to several autoantigens: ssDNA, double-stranded(ds)DNA and poly(ADP-ribose) but not RNA. PR4 and TH3 were used for preparation of rabbit anti-idiotype antisera. Inhibition studies demonstrated that the affinity purified rabbit anti-idiotype antisera were specific for their respective idiotype and that both Phen GL-I and ssDNA inhibited binding of idiotype to its anti-idiotype. PR4, but not TH3, was found to be similar but not identical to the 16/6 idiotype originally identified on a human monoclonal anti-DNA antibody derived from a patient with systemic lupus erythematosus (SLE).

Antibodies, Antinuclear↗

Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro.

Escherichia coli verotoxin (also known as Shiga-like toxin) has been implicated in the aetiology of the hemolytic uremic syndrome and hemorrhagic colitis. The glycolipid binding specificity of verotoxin purified from E. coli H30 and verotoxin cloned from bacteriophage H19B has been examined. Verotoxin from both sources binds specifically to globotriosyl ceramide containing the carbohydrate sequence galactose alpha 1-4galactose beta 1-4glucose-ceramide. Removal of the terminal galactose or substitution with N-acetylgalactosamine in beta 1-3 linkage deletes toxin binding activity. A ceramide trihexoside species, consistent with a globotriosyl ceramide structure was shown to be the major verotoxin-binding glycolipid of cultured vero cells which are routinely used to measure the cytotoxicity of toxin samples.

Bacterial Toxins↗

Mechanism of human platelet activation by endotoxic glycolipid-bearing mutant Re595 of Salmonella minnesota.

The mechanism through which human blood platelets interact with gram-negative bacteria with well-defined structural variations in endotoxic lipopolysaccharide was studied. Secretion of 14C-serotonin and aggregation of platelets separated from plasma proteins were observed on challenge with rough mutant Re595 of Salmonella minnesota possessing a glycolipid outer layer composed of Lipid A and 2-keto-3-deoxyoctonate (KDO) but lacking heptose phosphate in the core and O-polysaccharide in its outer portion. Both 14C-serotonin secretion and platelet aggregation were concentration-dependent, with a half-maximum response at the ratio of one bacterial colony-forming unit (CFU) to two platelets. The aggregation of human platelets induced by mutant Re595 was divalent cation-dependent and required secretion of ADP and fibrinogen from platelet storage granules because it was inhibited by chelators, by the ADP-splitting enzyme apyrase, and by monospecific antifibrinogen Fab fragments. The synthetic peptide analog of the platelet receptor recognition site on the gamma chain of fibrinogen, gamma 400-411, inhibited platelet aggregation induced by mutant Re595 (IC50 160 mumol/L), whereas serotonin secretion was unaffected. Tetrapeptide, RGDS, analogous to human fibrinogen alpha chain (alpha 572-575) and to the cell adhesion site of fibronectin, also inhibited aggregation induced by mutant Re595 (IC50 60 mumol/L). Secretion of 14C-serotonin was preceded by a very rapid phosphorylation of a platelet protein of mol wt 47,000, which is associated with protein kinase C activation. Myosin light chain (mol wt 20,000) was also phosphorylated. Both phosphoproteins were dephosphorylated while secretion was reaching maximum. Furthermore, release of 3H-arachidonic acid from platelet phospholipids and generation of thromboxane B2 via the cyclooxygenase pathway were observed. Inhibition of this pathway with acetylsalicylic acid (10(-4) mol/L) or indomethacin (5 X 10(-4) mol/L) reduced 14C-serotonin secretion and platelet aggregation. The role of Lipid A in the interaction of mutant Re595 with human platelets was deduced from the inhibitory effect of the Lipid A-binding protein present in Limulus amebocyte lysate. Likewise, polymyxin B, known to complex with Lipid A, was inhibitory. The reactivity of mutant Re595 toward platelets was attenuated by mild acid hydrolysis, during which KDO was dissociated from the glycolipid, and by alkaline hydrolysis, which breaks ester-linked fatty acids in Lipid A. In contrast to mutant Re595, strain S218 of S minnesota bearing "complete" endotoxic lipopolysaccharide did not induce secretion and aggregation of human platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗

Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. I. Evidence for tetrasaccharide structure of the oligosaccharide moiety.

Data from the literature indicate the presence, in the Mycobacterium kansasii wall, of a phenolic glycolipid called mycoside A. A tentative trisaccharide structure was proposed for the oligosaccharide part, whereas the aglycone was found to correspond to a phenol phthiocerol residue esterified by two mycocerosic acids. In the present work, structural information mainly arising from fast atom bombardment-mass spectrometry, tandem mass spectrometry, and 1H NMR of native and chemically degraded phenolic glycolipid indicates a tetrasaccharide structure for the inherent oligosaccharide. This structure is now determined as: 2,6-dideoxy-4-O-methyl-arabino-hexopyranosyl(1 alpha----3)2-O-methyl-4-O- acetylfucopyranosyl(1 alpha----3)2-O-methyl-rhamnopyranosyl(1 alpha----3)2-4-di- O-methylrhamnopyranosyl-1 alpha----phenolglycol. Evidence for the structure of the dideoxyhexose, unique in mycobacteria, is presented in the following paper (Fournié, J.-J., Rivière, M., Papa, F., and Puzo, G. (1987) J. Biol. Chem. 262, 3180-3184).

Antigens, Bacterial↗

Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. II. Presence of a novel dideoxyhexose.

A novel O-methyl-2,6-dideoxyhexose was isolated from the major phenolic glycolipid (previously called mycoside A) of Mycobacterium kansasii. Its molecular weight (162) was determined by gas chromatography-mass spectrometry analysis (chemical ionization with ammonia as reactant gas) of its underivatized reducing form. The methoxyl group was located by electron impact-mass spectrometry of its alditol acetate. The configuration was established by 1H NMR of its peracetylated derivative. The structure 2,6-dideoxy-4-O-methyl-arabino-hexopyranose is proposed for this new sugar. Evidence is also presented that the phenolic glycolipid previously called mycoside A is an antigen of M. kansasii since it reacts with rabbit antisera raised against whole M. kansasii.

Antigens, Bacterial↗

Inflammation induced by mycolic acid-containing glycolipids of Mycobacterium bovis (BCG).

Mycobacterium bovis (BCG) was submitted to lipid extraction involving gentle and drastic procedures. Neutral lipids, glycolipids as well as phospholipids were detected in the diethyl ether extract, whereas the chloroform and chloroform-methanol extracts contained mainly phospholipids. Arabinose mycolates, which represent firmly bound cell wall lipids, were detected in the acidified ether extract. The lipid extracts were adsorbed to charcoal particles and inoculated intravenously into mice. Histological analysis showed that the particles were trapped in the lung microcirculation and strong inflammatory reactions were induced around the charcoal particle coated lipids present in the diethyl ether, chloroform and acidified diethyl ether extracts. Fractionation of these inflammation-inducing extracts by column and preparative thin-layer chromatography led to the isolation of five active mycolic acid-containing glycolipids, identified as glycerol monomycolate, glucose monomycolate, arabinose monomycolate, trehalose monomycolate and trehalose dimycolate on the basis of physical, chemical and spectroscopic analyses. Inflammatory reactions similar to that observed after inoculation of live BCG were induced in the lungs by trehalose monomycolate, trehalose dimycolate or glucose monomycolate. However, the toxic reactions caused by glycerol monomycolate and arabinose monomycolate were characterized by an acute inflammatory process mainly due to the constant and massive presence of polymorphonuclear leukocytes.

Animals↗