Lewis blood group antigens defined by monoclonal anti-colon carcinoma antibodies

Monoclonal antibodies directed against human cancer cells were prepared by the murine hybridoma technique. These antibodies detect Lewis blood group antigens as determined by indirect solid-phase radioimmunoassay, hapten inhibition studies, and chromatogram binding assay. One monoclonal antibody is specific for the Lea terminal carbohydrate of Gal beta 1----3Glc NAc(4----1 alpha Fuc) beta 1----3LacCer. Five monoclonal antibodies react with the Leb terminal carbohydrate sequence of Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer, and four of these antibodies are highly specific for this glycolipid and do not react with other similar di- and monofucosylated glycolipids. One of the anti-Leb antibodies cross-reacts with blood group H glycolipid and has binding properties similar to those of the previously described antibody NS-10-17 [M. Brockhaus, J. L. Magnani, M. Blaszczyk, Z. Steplewski, H. Koprowski, K.-A. Karlsson, G. Larson, and V. Ginsburg (1981) J. Biol. Chem. 256, 13223-13225]. Two antibodies react with both the Lea and Leb antigens, though both bind preferentially to Leb.     

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.     

Lea-active heptaglycosylceramide, a hybrid of type 1 and type 2 chain, and the pattern of glycolipids with Lea, Leb, X (Lex), and Y (Ley) determinants in human blood cell membranes (ghosts). Evidence that type 2 chain can elongate repetitively but type 1 chain cannot

Two major glycolipids reactive with the monoclonal anti-Lea antibody have been isolated from human blood cell membranes. One component was identified as lactofucopentaosyl(II)ceramide and the other as a ceramide heptassaccharide with the structure described below: (formula; see text) The structure includes the Lea determinant (type 1 chain) linked to lactoneotetraosylceramide (type 2 chain); thus, it is regarded to be a hybrid between type 1 and 2 chain. In addition, a minor component having the thin-layer chromatographic mobility of a ceramide nonasaccharide, which was reactive to anti-Lea antibody, was detected. No other component with a thin-layer chromatographic mobility slower than the above components and reactive to the anti-Lea antibody was detected. In contrast, a series of slowly migrating glycolipids having X (Lex) determinant (Gal beta 1----4(Fuc alpha 1----3)GlcNAc) was detected. A similar series of long chain glycolipids having Y (Ley) determinant (Fuc alpha 1----2Gal beta 1----4(Fuc1----3)GlcNAc) was detected in human blood cells; in contrast, only one major Leb glycolipid was found with the mobility of a ceramide hexasaccharide. No glycolipid with a long carbohydrate chain composed exclusively of type 1 chain was detected. Thus, chain elongation may proceed through type 2 chain, but not through type 1 chain. Lea and X (Lex) haptens are distributed equally among blood group A, B, and O red blood cells, whereas the quantity of Leb and Y (Ley) haptens is much lower in A and B blood cells than in O blood cells.     

A new extended globoglycolipid carrying the stage specific embryonic antigen-1 (SSEA-1) determinant in mouse kidney

Two neutral glycolipids carrying the stage specific embryonic antigen-1 (SSEA-1) and SSEA-3 determinants, respectively, were purified from mouse kidney by a combination of column chromatographies and droplet counter-current chromatography. The structures of the glycolipids (GL-X and GL-Y) were determined by means of GLC, 1H-NMR spectroscopy, negative-ion fast atom bombardment mass spectrometry, a methylation study, and sequential degradation. GL-X was demonstrated to be galactosyl beta 1-3globotetraosylceramide, the structure of which had already been characterized to be that of SSEA-3 by Kannagi et al. [1983) J. Biol. Chem. 258, 8934-8942). GL-Y was a new glycolipid containing fucose, galactose, glucose, N-acetylgalactosamine, and N-acetylglucosamine in a molar ratio of 1:4:1:1:1. The methylation study results indicated that it contained 3 mol of terminal sugars composed of 2 mol of galactose and 1 mol of fucose with two branching points at N-acetylgalactosamine and N-acetylglucosamine. From the data obtained by 1H-NMR spectroscopy, mass spectrometry, and a binding assay using an anti-SSEA-1 monoclonal antibody (PM81) cloned by Ball et al. [1983) J. Immunol. 130, 2937-2941), we propose the structure of GL-Y to be Gal beta 1-4GlcNAc beta 1-6GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-ceramide. (sequence; see text) Fuc alpha 1 Gal beta 1 This is the first report on the isolation and characterization of a glycolipid carrying the SSEA-1 determinant on its globo-core structure.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.     

Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1-3)GalNAc-, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc- and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc- (Lewis X) terminal structures.

The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-electrospray ionization mass spectrometry. Apart from large, highly fucosylated structures with five to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues. Their structures have been determined as Fuc(alpha1-3)GalNAc(beta1-4)[ +/- Fuc (alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4) Glc-PA, Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-4) GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, and Fuc(alpha1-3) GalNAc(beta1-4)[ +/- Fuc(alpha1-2) +/- Fuc(alpha1-2)Fuc(alpha1-3)]Glc NAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA. The last structure exhibits a trifucosyl sidechain previously identified on the cercarial glycocalyx. These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates. To what degree these glycans contribute to the pronounced antigenicity of S. mansoni egg glycolipids remains to be determined. In addition, we have identified the compounds GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) GalNAc (beta1-4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(beta1-3)GalNAc(1-4)Gal(1-4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host.     

Y and blood group B type 2 glycolipid antigens accumulate in a human gastric carcinoma cell line as detected by monoclonal antibody. Isolation and characterization by mass spectrometry and NMR spectroscopy

A monoclonal antibody (mAb), BR55-2, was generated from mice immunized with MCF-7 human breast carcinoma cells. This mAb specifically detected glycolipids with the Y determinant Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)-beta 1----3Gal beta 1----4Glc beta 1----1 Cer and the Y-related B-active difucosylated determinant Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4GlcNAc(3----1 alpha Fuc) beta 1----3Gal beta 1----4Glc beta 1----1 Cer, but was not reactive with related monofucosylated glycolipids of type 2 chain (X-antigen, blood group H), type 1 chain (Lea antigen, blood group H and B) or with difucosylated type 2 and type 1 chain structures (A blood group antigen or blood group B and Leb, respectively). A series of glycolipids with Y and blood group B type 2 determinants were detected in human gastric adenocarcinoma cell line KATO III with mAb BR55-2 and with a previously characterized anti-blood group B mAb PA83-52 (Hansson, G. C., Karlsson, K.-A., Larson, G., McKibbin, J. M., Blaszczyk, M., Herlyn, M., Steplewski, Z., and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097). The isolated antigens were structurally characterized by mass spectrometry of permethylated and permethylated-reduced derivatives and by proton NMR spectroscopy. In a chromatogram binding assay, mAb BR55-2 and mAb PA83-52 detected minor components with slower mobility than the Y-6 and blood group B-7-type 2 structures. The detection of a B type 2 determinant is the first chemical evidence for the presence of an autologous difucosyl blood group B type 2 antigen in human adenocarcinoma cells.     

The hapten structure of a developmentally regulated glycolipid antigen (SSEA-1) isolated from human erythrocytes and adenocarcinoma: a preliminary note

Glycolipid antigen reacting to the monoclonal antibody directed to the developmentally regulated antigen SSEA-1 was isolated from human erythrocytes and colonic adenocarcinoma. The antigens have the Le^x (Galβl→4[Fucα]→3]GlcNAcβl→R) or Le^y (Fucαl→2Galβl→4[Fucαl→3]GlcNAcβl→R) structure at the termini of the branched polylactosaminolipid. In addition, a novel polyfucosyl structure locating exclusively at the internal GlcNAc was detected in the tumor antigen. The antibody reacts with a simple monovalent Le^x glycolipid (Galβl→4[Fucαl→3]GlcNAcβl→3Galβl→4Glcβl→Cer) previously isolated from colonic carcinoma when presented at a high density on liposomes. The antibody therefore may react to the bivalent or multivalent Le^x or Le^y structure.     

Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.     

Primary structure of human milk nona- and decasaccharides determined by a combination of fast atom bombardment mass spectrometry and1H-/13C-nuclear magnetic resonance spectroscopy. Evidence for a new core structure,iso-lacto-N-octaose

The structure of a nonasaccharide and of two decasaccharides isolated from human milk has been investigated by using methylation, fast atom bombardment mass spectrometry and 1H-/13C-nuclear magnetic resonance spectroscopy. The structures of these oligosaccharides were: trifucosyllacto-N-hexaose; Fuc alpha 1-2Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4Glc, difucosyllacto-N-octaoses; Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc and Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Fuc alpha 1-3 Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc. The two decasaccharides possess a new type of core structure proposed to be named iso-lacto-N-octaose.     

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)     

Assignment of the1H- and13C-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series

The assignment of the 13C- and 1H-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series was obtained from homonuclear and heteronuclear correlation spectroscopy. These analyses were performed on the following compounds: 1. Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 2. NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 3. Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 4. NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 5. NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc; 6. Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 7. Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; 8. NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc.     

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.     

Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine

Many monoclonal antibodies that react with the lacto-N-fucopentaose III (LNF III) antigenic determinant, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc, have been described recently. The terminal trisaccharide of this determinant, fucosyllactosamine, is present on glycolipids and glycoproteins and on the surface of granulocytes, monocytes, and other cells. To study the structural and genetic diversity of these antibodies, syngeneic anti-idiotypic monoclonal antibodies were produced in BALB/c mice against PMN 6, a monoclonal antibody directed against this sequence. Anti-idiotypic antibodies 6B1 and 6C4 reacted with 50% of a panel of 20 anti-LNF III monoclonal antibodies, whereas 6A3 reacted strongly only with PMN 6. This indicates that the determinants recognized by 6C4 and 6B1 represent major cross-reactive idiotopes of this family of antibodies. The binding of idiotypic antibodies to a glycolipid bearing this antigenic determinant was completely inhibited by the three anti-idiotypic antibodies, 6A3, 6B1, and 6C4. The idiotopes could be demonstrated on the heavy chain of the monoclonal antibodies by an antibody transfer technique when mild reducing conditions were employed, but a high concentration of reducing agent destroyed the idiotypic determinants. This suggests that the anti-idiotypic antibodies recognize conformational structures expressed on the heavy chain molecules. The binding of 18 monoclonal antibodies to two glycolipid antigens and to a fucosyllactosamine-bovine serum albumin conjugate was compared. Antibodies that possessed the 6C4 cross-reactive idiotope bound to fucosyllactosamine-bovine serum albumin more weakly than idiotype-negative antibodies (p = 0.001). This suggests that the 6C4-positive antibodies might represent germline structures.     

Complete analysis of the1H- and13C-NMR spectra of four blood-group A active oligosaccharides

The fully assigned 1H and 13C-NMR spectra of four group A oligosaccharides by use of multiple-relayed, coherence-transfer chemical-shift-correlated spectroscopy (multiple-RELAY-COSY) and 1H-/13C-correlation spectroscopy are reported. These analyses were performed on the following compounds: III-A; GalNAc alpha 1-3[Fuc alpha 1-2]Gal: VI-A; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal: VII-A-1; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-1Glycerol: VII-A-2; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc.     

Novel fucolipids accumulating in human adenocarcinoma. II. Selective isolation of hybridoma antibodies that differentially recognize mono-, di-, and trifucosylated type 2 chain

A series of glycolipids having the X determinant (Gal beta 1----4 [Fuc alpha----3]GlcNAc) at the terminus and a fucosyl alpha 1----3 residue at the internal GlcNAc residue have been isolated and characterized from tumor tissues (Hakomori, S., Nudelman, E., Levery, S.B., and Kannagi, R. (1984) J. Biol. Chem. 259, 4672-4680. A series of monoclonal antibodies that differentially recognize glycolipids with mono-, di-, and trifucosylated type 2 chain have been isolated and characterized. The antibody FH4 shows a remarkable preferential reactivity towards di-/or trifucosylated type 2 chain, i.e. it does not react with monofucosylated structures, including lactofucopentaosyl (III) ceramide (III3FucnLc4), monofucosyl neolactonorhexaosylceramide (y2, V3FucnLc6), and monofucosyl neolactonoroctaosylceramide (Z1, VII3FucnLc8), but reacts well with di- and trifucosylated type 2 chain structures such as difucosyl neolactonorhexaosylceramide (III3V3Fuc2nLc6) and trifucosyl neolactonoroctaosylceramide (III3V3VII3Fuc3nLc8). Two other monoclonal antibodies, FH5 and ACFH18, preferentially react with trifucosylated type 2 chain structure (III3V3VII3Fuc3nLc8), although cross-reactivity with difucosylated type 2 chain (III3V3Fuc2nLc6) was observed. They showed a minimal cross-reaction with monofucosylated type 2 chain. In contrast, the antibody FH1 does not react with III3FucnLc4 but reacts with V3FucnLc6, III3V3Fuc2nLc6, and III3V3VII3Fuc3nLc8. Two monoclonal antibodies, FH2 and FH3, do not discriminate among various glycolipids having fucosylated type 2 chain, and their reactivities are essentially similar to previously established antibodies directed to the X determinant, such as anti-SSEA-1, WGHS 29, VEP8 and 9, My-1, etc. This series of antibodies will be useful to detect the specific type of glycolipid with fucosylated type 2 chain accumulating in human cancer and in undifferentiated cells.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.     

Biosynthesis of the cancer-associated sialyl-Lea antigen

A cancer-associated glycolipid antigen defined by monoclonal antibody 19-9 has the structure NeuAc alpha 2-3Gal Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer. We have (formula; see text) studied its biosynthesis by testing the capacity of a crude microsomal fraction of SW 1116 cells to catalyze the addition of fucosyl or sialyl residues from GDP-fucose or CMP-sialic acid to glycolipid or oligosaccharide precursors. When the tetrasaccharide NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LSTa) is incubated with GDP-[14C]fucose and SW 1116 microsomes, a 14C-labeled oligosaccharide is formed that can be separated from the incubation mixture on an affinity column containing antibody 19-9 bound to protein A-Sepharose. The product migrates slower than LSTa when analyzed by paper or thin-layer chromatography. After treatment with neuraminidase, it co-migrates with the pentasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (formula; see text) (LNF II) in both chromatographic systems. Similar experiments demonstrate that SW 1116 microsomes catalyze the addition of a sialyl residue to the tetrasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc to form LSTa. However, when LNF II is incubated with CMP-[14C]sialic acid and SW 1116 microsomes, no 19-9-active product is detected by affinity chromatography or by paper or thin-layer chromatography. Results using glycolipid precursors are consistent with these findings and also demonstrate the presence of the Lewis fucosyltransferase in SW 1116 cells. Thus, the biosynthesis of the sialyl-Lea antigen proceeds by addition of sialic acid to a type 1 precursor chain by a sialyltransferase, followed by addition of fucose by the Lewis fucosyltransferase.