Immunochemistry of I/i-active oligo- and polyglycosylceramides from rabbit erythrocyte membranes. Determination of branching patterns of a ceramide pentadecasaccharide by 1H nuclear magnetic resonance

1H NMR spectra of the ceramide hexasaccharide obtained after the removal of the terminal alpha-Gal and subterminal beta-Gal residues from the ceramide decasaccharide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc (beta 1-6)]Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, showed that terminal and internal GlcNAc residues are differentiated by their chemical shifts. This finding enabled us to determine the primary structure of the title compound as Gal(alpha 1-3)Gal(beta 1-4)GlcNAc (beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-6)]Gal(beta 1-4)GlcNAc (beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-6)]Gal(beta 1-4)GlcNAc (beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer. Alternative branching of this oligosaccharide chain was excluded since the removal of all terminal alpha-Gal and penultimate beta-Gal residues yielded a ceramide nonasaccharide containing one terminal and two internal 1----3-linked GlcNAc residues, as well as two terminal 1----6-linked GlcNAc units. The intermediate degradation products of the ceramide deca- and pentadecasaccharides , viz. the ceramide octa- and dodecasaccharide , obtained by the removal of alpha-Gal residues only, as well as the linear ceramide heptasaccharide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3) Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, and ceramide hexasaccharide, Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc (beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, were also investigated. The usefulness of the glycosylation-induced chemical shifts is discussed.     

Characterization of the lacto series of gangliosides, especially of disialo-lacto-N-norhexaosyl ceramide isolated from adult bovine nasal cartilage

A disialosylganglioside was isolated from adult bovine nasal cartilage, and its structure was determined by analysis of sugar composition, permethylation analysis, exoglycosidase treatment, and mild acid hydrolysis. The structure of this ganglioside was identified as disialo-lacto-N-norhexaosyl ceramide, NeuNAc(alpha 2-8)NeuNAc-(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(1-4)Glc(1-1)Cer. Furthermore, we also isolated from this cartilage gangliosides whose structures were presumed to be monosialo-lacto-N-norhexaosyl ceramide, and mono- and disialo-lacto-N-neotetraosyl ceramide. The major fatty acids of the four gangliosides isolated were palmitic, stearic, behenic and lignoceric acids. The predominant long chain bases were sphingenine, heptadecasphingenine and hexadecasphingenine.     

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.     

Specific inhibition of macrophage migration inhibition factor by fucosylated glycolipid RM.

The effects of glycolipids on the interaction of the MIF (migration inhibition factor) with rat macrophages were examined using a migration inhibition assay system. MIF activity was specifically blocked by fucosylated Glycolipid RM [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-3GalNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide, (1978) J. Biochem. 83, 85-90], but not by Cytolipin R, hematoside, or blood group B active glycolipid [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide]. Inhibition of MIF activity was proportional to the concentration of Glycolipid RM. These findings suggest that Glycolipid RM acts as a receptor for MIF.     

Characterization of the binding of propionibacterium granulosum to glycosphingolipids adsorbed on surfaces. An apparent recognition of lactose which is dependent on the ceramide structure

The binding properties of a strain of Propionibacterium granulosum derived from human skin was investigated with reference to glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells using externally (125I) and metabolically [( 35S]methionine) labeled bacteria. Binding was found to lactosylceramide (LacCer; Gal beta 1-4Glc beta 1-Cer) but not to glycolipids lacking the lactose sequence (i.e. Glc beta 1-Cer, Gal beta 1-Cer or Gal alpha 1-4Gal beta 1-Cer). In microtiter wells, binding occurred at 50 ng and became half-maximal at the theoretical value for a monomolecular layer of LacCer (i.e. 100-200 ng/well). The bacteria also bound to glycolipids with various substitutions (e.g. GalNAc beta 1-4, Gal beta 1-3GalNAc beta 1-4, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4, Gal alpha 1-3, GlcNAc beta 1-3, Gal beta 1-3GlcNAc beta 1-3, Gal beta 1-4GlcNAc beta 1-3, and Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3) at the Gal of LacCer, although only those species with GalNAc beta 1-4 or Gal beta 1-3GalNAc beta 1-4 were as active as LacCer itself. Glycolipids with other additions (e.g. Gal alpha 1-4 and NeuAc alpha 2-3) were negative. For unsubstituted LacCer, the binding required either a trihydroxy base or 2-hydroxy fatty acid, specifying the epithelial type of ceramide; LacCer composed of a dihydroxy base and nonhydroxy fatty acid was negative. This is interpreted as indicating that the proper presentation of the binding epitope depends on the ceramide structure. The relevance of this to biological membranes has not yet been established. Neither free lactose (up to 20 mg/ml) nor lactose-bovine serum albumin (5 mg/ml) prevented the binding of bacteria to LacCer, two facts that support the solid-phase binding data demonstrating a low affinity binding and the crucial importance of a particular lactose epitope.     

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(beta1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity

Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal(alpha1-4)Gal(beta1-4)Glc(1-1)ceramide and Gal(alpha1-3)Gal(beta1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc(alpha1-3)GalNAc(beta1-3/4)Gal(alpha1-4/3)Gal(beta1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal alpha-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(beta1-6)Gal(beta1-4)Glc(1-1)ceramide, Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide and Gal(beta1-6)Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(beta1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(beta1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

Studies on the chemical structure of neutral glycosphingolipids in eggs of the sea hare, Aplysia juliana.

Six neutral glycosphingolipids (GL-1-GL-6) were obtained from eggs of the sea hare (Aplysia juliana) and were characterized by FABMS, 1H-NMR, partial acid hydrolysis, methylation studies and GC analysis of the component sugars, fatty acids and long-chain bases. The following structures were determined to be Glc beta 1-1Cer (89%) and Gal beta 1-1Cer (11%) for GL-1, Glc beta 1-1Cer (47%) and Gal beta 1-1Cer (53%) for GL-2 having hydroxy fatty acids in the ceramide moiety, Gal beta 1-4Glc beta 1-1Cer for GL-3, Fuc alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-4, Gal alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-5 and GalNAc alpha 1-3(Gal alpha 1-2)Gal beta 1-4Glc beta 1-1Cer for GL-6. The fatty acid composition of each glycosphingolipid, except for GL-2, which contained 2-hydroxypalmitic acid, consisted of mostly saturated C16-C20 acids, especially palmitic acid and stearic acid. The long-chain bases of all glycosphingolipids consisted mainly of branched nonadeca-4-sphingenine and octadeca-4-sphingenine. GL-6, which was one of the major glycosphingolipids, may be a precursor of a series of phosphonoglycosphingolipids which have been isolated from the skin of A. kurodai.     

Glycosphingolipids in insects. Chemical structures of ceramide monosaccharide, disaccharide, and trisaccharide from pupae of Calliphora vicina (Insecta: Diptera)

The presence of glycosphingolipids in the pupae of the blowfly, Calliphora vicina, was established. The thin layer chromatographic pattern of the total neutral glycolipids revealed the presence of more than 13 components, the major one being ceramide monohexoside. By the use of high performance liquid chromatography, the three simplest components were isolated and their chemical structures determined: Glc(beta 1-1)Cer, Man(beta 1-4)-Glc(beta 1-1)Cer [with minor component Gal(beta 1-4)Glc(beta 1-1)Cer] and GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)-Cer. The ceramide composition of the parent insect glycosphingolipids is dominated by the 20:0 fatty acid, arachidic acid, and the sphingoid tetradecasphing-4-enine.     

GalNAc beta 1----3 terminated glycosphingolipids of human erythrocytes

Nonacid glycosphingolipids with 4 to 10 sugar residues isolated from pooled erythrocytes of blood group O donors have been efficiently separated as peracetylated derivatives on silicic acid. This procedure enabled a quantitative estimate of individual compounds and also revealed several GalNAc beta 1----3 terminated structures. The structural characterization of these glycolipids with 1H-NMR spectroscopy, direct inlet mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry identified the compounds as GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl sphingosine and GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl phytosphingosine, GalNAc beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 ceramide, and GalNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.     

Carbohydrate and hydrophobic-carbohydrate recognition sites (CARS and HY-CARS) in solubilized glycosyltransferases

Six different glycosyltransferases that are active with glycosphingolipid substrates have been purified from Golgi-membranes after solubilization with detergents. It appears that GalT-4(UDP-Gal:GlcNAc-R1 beta 1-4GalT), GalNAcT-2(UDP-Gal:Gal alpha-R2 beta 1-3GalNAcT) and FucT-2(GDP-Fuc:Gal beta GlcNAc-R3 alpha 1-2FucT) are specific for oligosaccharides bound to ceramide or to a protein moiety. These are called CARS (carbohydrate recognition sites) glycosyltransferases (GLTs). On the other hand, GalT-3(UDP-Gal:GM2 beta 1-3GalT), GalNAcT-1(UDP-GalNAc:GM3 beta 1-4GalNAcT) and FucT-3 (GDP-Fuc:LM1 alpha 1-3FucT) recognize both hydrophobic moieties (fatty acid of ceramide) as well as the oligosaccharide chains of the substrates. These GLTs are called HY-CARS (hydrophobic and carbohydrate recognition sites). D-Erythro-sphingosine (100-500 microM) modulates the in vitro activities of these GLTs. Modulation depends on the binding of D-sphingosine to a protein backbone, perhaps on more than one site and beyond transmembrane hydrophobic domains. Control of GLTs by free D-sphingosine was suggested with the concomitant discovery of ceramide glycanase in rabbit mammary tissues. The role of free sphingosine as an in vivo homotropic modulator of glycosyltransferases is becoming apparent.     

Three-dimensional structure of the oligosaccharide terminus of globotriaosylceramide and isoglobotriaosylceramide in solution. A rotating-frame NOE study using hydroxyl groups as long-range sensors in conformational analysis by 1H-NMR spectroscopy

Spatial structures of the oligosaccharide parts of globotriaosylceramide, Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer (Cer = ceramide) and isoglobotriaosylceramide, Gal(alpha 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer were investigated in (C2H3)2SO solution by means of laboratory and rotating frame NOE, hydroxyl protons being used as long-range sensors defining the distance constraints. Both oligosaccharides were found to exist in more than one conformation interconverting rapidly on the NMR time scale. The conformation of the Gal(alpha 1-4)Gal(beta 1-4)Glc beta trisaccharide dissolved in 2H2O appeared to be the same as that of the corresponding part of the glycosphingolipid in (C2H3)2SO solution.     

Structure of the human erythrocyte blood group P1 glycosphingolipid.

A glycosphingolipid with blood group P1 activity was extracted from an acetone powder of human erythrocyte stroma with chloroform-methanol. It was purified by chromatography on columns of silicic acid and by preparative thin-layer chromatography of the fully acetylated and deacetylated glycolipid. The purified glycolipid contained galactose, N-acetylglucosamine, and glucose in a molar ratio of 3:1:1. Treatment of the P1 glycolipid with fig alpha-galactosidase released a single galactosyl residue and destroyed the blood group activity, and the alpha-galactosidase product had the same chromatographic mobility as paragloboside. Substitution sites on the neutral sugars of the P1 glycolipid and the alpha-galactosidase product were established by identification of methylated alditol acetates, and substitution on N-acetylglucosamine was determined by identification of methyl glycoside derivatives. The terminal nonreducing disaccharide of the P1 glycolipid is Gal(alpha, 1 leads to 4)Gal. N-Acetylglucosamine was identified as the next sugar in sequence by mass spectrometric analysis of the permethylated P1 glycolipid. On the assumption that the glucose residue is linked to ceramide, we propose the following structure for the P1 glycolipid: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-NAc(beta, 1 leads to 4)Glc-Cer.     

Biosynthesis in vitro of SA-Lex and SA-diLex by alpha 1-3 fucosyltransferases from colon carcinoma cells and embryonic brain tissues.

The sialyl-fucosyl-lactosamine-epitope present in sialyl (SA)-Lex (NeuAc alpha 2-3Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta 1-3Gal beta 1-4Glc-Cer), a carcinoembryonic antigen, has been recognized recently as a ligand for the binding of leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) to myeloid and tumour cell surfaces. We have recently detected the presence of an alpha 1-3 fucosyltransferase (FucT-3) activity in both embryonic chicken brain (ECB) and human colon carcinoma cells (Colo-205) which catalyses the biosynthesis in vitro of SA-Lex and SA-diLex. Fucosyltransferase activities from both sources are stimulated in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Co2+ and Fe2+), although absolute metal requirement is not observed. Substrate specificity studies with this partially purified (ECB, 3000-fold; Colo-205, 100-fold) novel FucT-3 indicate the preference for terminally sialyl-substituted glycolipid acceptors, as observed by the lower Km values when sialyl-neolactotetraosyl ceramide, LM1, (Neu-Gc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4 Glc-Cer; Km = 0.048 mM) and sialyl-norhexaosylceramide, NeuGc-nLc6, (Neu-Gc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer; Km = 0.032 mM) were used as substrates. Fucosyltransferase from Colo-205 requires the presence of the acyl group of the ceramide moiety and an acetyl group on glucosamine in the acceptor glycolipid since lyso-LM1 was found to be completely inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel ceramide trihexoside from the eggs of the sea urchin, Hemicentrotus pulcherrimus.

Glucosylceramide (Glc beta 1-1Cer) and a novel ceramide trihexoside (Gal beta 1-6Gal beta 1-6Glc beta 1-1Cer) were purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, chromic acid oxidation, enzymatic hydrolysis, enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ceramide trihexoside has a novel carbohydrate structure, and its core structure, Gal beta 1-6Glc, is also novel. The ceramide moieties of these glycolipids are almost identical. Two fatty acids, 22:1 and 22h:1, constitute more than 80% of the total acids. Long-chain bases are all phytosphingosine, approximately 90% of which is n-t18:0. The finding of melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) from the eggs of another sea urchin species [Kubo, H. et al. (1988) J. Biochem. 104, 755-760] and the present finding of the novel ceramide trihexoside suggest that there are a variety of unique sugar structures in sea urchin glycosphingolipids.     

Heterophile antibodies to rabbit erythrocytes in human sera and identification of the antigen as a glycolipid

Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer.     

Reactivity of mouse monoclonal antibody M2590 against B16 melanoma cells with chemically synthesized GM3 ganglioside

A mouse (C57BL/6) monoclonal antibody M2590, which is established against syngeneic melanoma B16 cells, reacted with chemically synthesized GM3. NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1' ceramide (24:0/d 18:1), but not with its stereoisomer, NeuAc beta 2-3Gal beta 1-4Glc beta 1-1' ceramide (24:0/d 18:1).     

Identification of erythrocyte Gal alpha 1-3Gal glycosphingolipids with a mouse monoclonal antibody, Gal-13

The Gal alpha 1-3Gal structural determinant has been found to have a unique distribution in mammals. Although this determinant is abundantly expressed by erythrocytes and nucleated cells of many mammals, it has not been detected in human cells. However, our previous studies (Galili, U., Rachmilewitz, E. A., Peleg, A., and Flechner, I. (1984) J. Exp. Med. 160, 1519-1531; Galili, U., Clark, M. R., and Shohet, S. B. (1986) J. Clin. Invest. 77, 27-33) have suggested that this epitope is present in small amounts and may be involved in immune-mediated destruction of senescent human erythrocytes. To have a means for exploring this possibility and for studying the species and tissue distribution of this epitope we have raised a monoclonal antibody (Gal-13) which specifically binds to glycoconjugates with a nonreducing terminal Gal alpha 1-3Gal disaccharide. Mice were immunized with rabbit erythrocytes, which express an abundance of glycoconjugates with Gal alpha 1-3Gal epitopes. Clones were screened with a solid-phase binding assay (enzyme-linked immunosorbent assay) for antibodies which bound to ceramide pentahexoside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3-Gal beta Gal beta 1-4Glc1-1Cer) but not to ceramide trihexoside (Gal alpha 1-4Gal beta 1-4Glc1-1Cer). Gal-13 bound to a number of neutral glycosphingolipids from rabbit and bovine erythrocytes. These glycosphingolipids have previously been shown to be a family of linear and branched polylactosamine structures, which have non-reducing terminal Gal alpha 1-3Gal epitopes. The antibody did not bind to the human blood group B glycolipid, Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc1-1Cer, and, therefore, branching at the penultimate galactose blocks Gal-13 binding. However, after removal of the fucose from the B antigen Gal-13 recognized the resulting derivative. Other Gal alpha 1-3Gal glycosphingolipids with an isogloboside or globoside core structure were not recognized by Gal-13 suggesting that the antibody binds to Gal alpha 1-3Gal carried by a lactosamine core structure. Gal-13 has been used to demonstrate that the Gal alpha 1-3Gal ceramide pentahexoside has been evolutionarily conserved in red cells of animals up to the stage of New World monkeys but is not found in Old World monkey red cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycosphingolipids of porcine pancreas

Neutral and acidic glycosphingolipids were purified from porcine pancreas by chromatography on columns of DEAE-Sephadex and Iatrobeads. The chemical structures of the purified glycolipids were determined by carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, NMR and IR. The major glycolipid of porcine pancreas was Gal(alpha,1-4)Gal(beta,1-)ceramide. Gangliosides GM3 and GD3 were major acidic components and galactosylceramide 3-sulfate was also found.