The glycosphingolipid composition of the placenta of a blood group P fetus delivered by a blood group Pk1 woman and analysis of the anti-globoside antibodies found in maternal serum

To further define the molecules that may mediate spontaneous abortion due to maternal-fetal blood group incompatibility within the P blood group system, we have examined the fine specificities of maternal antibodies and the glycolipid antigens from the placenta of a P infant born to a Pk1 mother. Maternal antibodies obtained during therapeutic plasmapheresis were analyzed to determine their reactivities with placental glycolipid extracts on thin-layer plates. Second antibodies specific for IgM, IgG, and IgA revealed immunoglobulins of all of these classes strongly reactive with one major placental glycolipid that comigrates with globoside. GC/MS analysis confirmed that the major P-active pentaglycosylceramide of placenta has the same structure as that previously shown for the P antigen of red blood cells: GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. Serum antibodies partially purified by affinity chromatography on globoside-octyl-Sepharose specifically recognize glycolipids that contain terminal GalNAc beta 1-3Gal . . . residues and also recognize the same sequence as an internal determinant in some, but not all, glycolipids with extended globoside core regions. Thus, in the blood group P incompatible fetus, the major P antigen present in placenta has the same carbohydrate structure as the P antigen present in fetal and adult erythrocytes and might be a target for the maternal immune system.     

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.     

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.     

Anti-alpha-galactosyl antibodies recognizing epitopes terminating with alpha1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.     

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)     

Characterization of N-glycolyneuraminic acid-containing glycosphingolipids from a Marek's disease lymphoma-derived chicken cell line, MSB1, as tumor-associated heterophile Hanganutziu-Deicher antigens

Heterophile, Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid-containing glycosphingolipids (GSLs) were detected as tumor-associated foreign antigens of a Marek's disease lymphoma-derived cell line, MSB1, by enzyme-immunoassay with chicken antibody against N-glycolylneuraminyl-lactosylceramide (anti-NeuGc-LacCer). At least three species of HD antigen-active GSLs were detected by two-dimensional thin-layer chromatography (TLC) combined with enzyme-immunoassay. The reactivities of the GSLs with anti-NeuGc-LacCer, their behaviors on two-dimensional TLC and the results of an endo-beta-galactosidase digestion study indicated that these three GSLs were NeuGc-LacCer (NeuGc alpha 2-2Gal beta 1-4Glc-Cer), NeuGc-nLcOse4Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and NeuGc-nLcOse6Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer).     

Carbohydrate analysis of chicken heart glycolipids

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).     

Globotetraosylceramide is recognized by the pig edema disease toxin

The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.). Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer). Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin. Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized. This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor. The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.     

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.     

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.     

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.     

Monoclonal antibodies detecting different epitopes on the Forssman glycolipid hapten

Two hybridomas, derived by fusing mouse myeloma cells with spleen cells from a rat immunized with mouse mammary tumors, have been shown to produce antibodies that recognize cell surface antigens on mesenchymal cells in a variety of tissues. Evidence presented in this report suggests that these antibodies detect overlapping epitopes on the Forssman glycolipid hapten (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). One antibody (33B12) reacts with the terminal sugar sequence GalNAc alpha 1-3GalNAc and is specific for Forssman. The other antibody (117C9) recognizes the internal sugar sequence GalNAc beta 1-3Gal. The terminal sugar sequence GalNAc beta 1-3Gal in globoside, as well as the internal sugar sequence GalNAc beta 1-4Gal in asialo-GM1, is not recognized as an antigenic determinant by 117C9. Nevertheless, the 117C9 antibody does not react exclusively with the Forssman antigen. In a lipid extract fractionated by Folch partition of mouse mammary tumors, the antibody also detects other glycolipids.     

The occurrence of glycosphingolipids containing mannose in the sea-water bivalve, Meretrix lusoria (Hamaguri)

Total neutral and acidic glycosphingolipids were prepared from whole tissues of the sea-water bivalve, Meretrix lusoria, and the former preparation was further fractionated into subgroups by silicic acid column chromatography. The fractions obtained as mono-(ceramide monosaccharide, CMS), di-(CDS) and triglycosylceramides (CTS) were characterized by thin-layer chromatography, partial hydrolysis with exoglycosidases, methylation studies, CrO3 oxidation, and GLC analysis of the component sugars, fatty acids and long-chain bases. The following structures are proposed: Gal-Cer and Glc-Cer for CMS, Gal(beta 1----4)Glc-Cer and Man(beta 1----4)Glc-Cer (MlOse2Cer) for CDS, Man(alpha 1----3)Man(beta 1----4)Glc-Cer (MlOse3Cer) and Gal(alpha 1----3)Man(beta 1----4)Glc-Cer (II3 alpha Gal-MlOse2Cer) for CTS. To our knowledge II3 alpha Gal-MlOse2Cer has not previously been reported. The fatty acid composition of CMS, CDS, and CTS consisted almost entirely of saturated C16-C24 acids with large amounts of 2-hydroxypalmitic acid and 2-hydroxystearic acid. The long-chain bases consisted of 4-sphingenine and 4,8-sphingadienine. More complex neutral glycolipids than CTS, as well as an acidic glycolipid, were examined by TLC and GLC of the constituent sugars, and an immunochemical technique.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides.

Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.     

Human erythrocyte P and Pk blood group antigens: identification as glycosphingolipids

The human erythrocyte P blood group system consists of three known antigens, P₁, P and P^k. We have identified the P antigen as the glycosphingo-lipid globoside, βGalNAc(1→3)αGal(1→4)βGal(1→4)Glc-cer, and the P^k antigen as ceramide trihexoside, αGal(1→4)βGal(1→4)Glc-cer. These data suggest, in contrast to previous hypotheses, that the P^k antigen is a biosynthetic precursor of P, and that neither P nor P^k is a precursor of P₁. These findings also provide an explanation for the apparent recessive inheritance of the P^k antigen, and for the nature of the biochemical abnormality in individuals of the rare P^k and p phenotypes.     

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.     

Chemical structure of carcinoma ganglioside antigens defined by monoclonal antibody C-50 and some allied gangliosides of human pancreatic adenocarcinoma

A hybridoma, C-50, obtained by fusion of mouse myeloma cells with spleen cells from a mouse immunized with cells from the colorectal carcinoma cell line COLO 205, produced antibodies that detected ganglioside antigen in human adenocarcinomas in many organs. The major ganglioside antigen fraction isolated from liver metastases of a pancreatic adenocarcinoma, behaving as a homogenous band on thin-layer chromatography, consisted of three different gangliosides. One of them, A (25%), had the same carbohydrate structure as the ganglioside antigen defined by monoclonal antibody 19-9, NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4Glc-Cer(Fuc-3'-isoLM1) Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H. and Ginsburg, V. (1982) J. Biol. Chem. 257, 14365-14369). The major ganglioside, B (60%), was the isomeric hexasaccharide ganglioside (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3-Gal beta 1-4Glc-Cer(Fuc-3'-LM1) and the third ganglioside, C, was 6'-LM1, NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer (15%). Ganglioside B, isolated from human kidney, did not react with the C-50 MAb. Based on this result and on studies of COLO 205 cell induced tumours where the ganglioside antigen fraction only consisted of A, it is suggested that the C-50 MAb defines an antigen determinant present in A.     

Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.     

The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Accumulation of a globo-series glycolipid having Gal alpha 1-3Gal in PC12h pheochromocytoma cells

In a previous paper, we reported the presence of globoside as a major neutral glycolipid in PC12 pheochromocytoma cells [Ariga, T., Macala, L. J., Saito, M., Margolis, R. K., Greene, L. A., Margolis, R. U., & Yu, R. K. (1988) Biochemistry 27, 52-58]. Recently, we found that subcloned PC12h cells accumulated another unusual neutral glycolipid. In order to characterize this glycolipid, PC12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated two major neutral glycolipids, which were purified by Iatrobeads column and preparative thin-layer chromatographies. One of the glycolipids was found to be globoside, and the other had a globotriaosyl structure with an additional terminal Gal alpha 1-3 residue. Its structure was determined by fast atom bombardment mass spectrometry, two-dimensional proton nuclear magnetic resonance spectrometry (2D NMR), permethylation study, sequential degradation with exoglycosidase, and mild acid hydrolysis to be Gal(alpha 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1')Cer.