Monoclonal antibodies defining blood group A variants with difucosyl type 1 chain (ALeb) and difucosyl type 2 chain (ALey).

Three hybridomas secreting monoclonal antibodies, HH1, HH2, and HH3, defining different difucosyl A structures (ALeb or ALey), have been established. Antibody HH1 (IgG2a) reacts specifically with the difucosyl A structure irrespective of a type 1 or type 2 chain, while antibody HH2 (IgG3) reacts exclusively with the difucosyl type 2 chain A (ALey) and does not react with the difucosyl type 1 chain or monofucosyl type 2 chain. Antibody HH3 (IgG2a) reacts exclusively with the difucosyl type 1 chain A (ALeb) and does not react with the monofucosyl type 1 chain A or mono- and difucosyl type 2 chain A. These hybridoma antibodies were obtained by immunization of mice with purified glycolipid antigens and were selected by their reactivity with the specific glycolipid structures. These antibodies, together with previously established monoclonal antibody AH-21, specific for monofucosyl type 1 chain A, and monoclonal antibody TH-1, specific for type 3 chain A, are extremely useful to define blood group A variants present in cells and tissues.

Blood group A determinants with mono- and difucosyl type 1 chain in human erythrocyte membranes.

Application of a monoclonal antibody defining monofucosyl type 1 chain A (AH21) revealed the presence of a glycolipid having the same thin-layer chromatography mobility as Aa but showing a clear reactivity with AH21. This glycolipid was detectable in Lea-b- erythrocytes but not in Lea+b- or Lea-b+ erythrocytes. Another monoclonal antibody defining difucosyl type 1 chain A (HH3) detected the presence of a glycolipid component reacting with this antibody in Lea-b+ erythrocytes but not in Lea+b- or Lea-b- erythrocytes. The component defined by monoclonal antibody AH21 and that defined by HH3 were isolated and characterized by 1H NMR spectrometry and methylation analysis as having the structures (Formula: see text) The 1H NMR spectra of these glycolipid antigens were characterized by resonances for anomeric protons that are identical with those of glycolipids with type 1 chain previously isolated but distinctively different from those of type 2 chain analogues. Resonances reflecting ceramide composition are characteristic for these antigens from human erythrocytes and are distinguishable from those of the same antigen from other sources.

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.

Chemical characterization of a blood group H type pentaglycosylceramide of human small intestine.

A blood group H type pentaglycosylceramide was isolated in relatively large amounts from human adult small intestine (52 mg from one individual) and human meconium (fetal origin). The structure was made likely by mass spectrometry and NMR spectroscopy of non-degraded permethylated and permethylated-LiAlH4-reduced glycolipid and by degradation to be Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. The ceramide was composed mainly of phytosphingosine and 2-hydroxy 16-24 carbon fatty acids. This novel type 1 chain species (Gal beta 1 leads to 3GlcNAc) was not accompanied by the type 2 chain isomer (Gal beta 1 leads to 4GlcNAc) which in contrast is the sole species in human erythrocyte and dog small intestine.

Mouse monoclonal antibodies against human cancer cell lines with specificities for blood group and related antigens. Characterization by antibody binding to glycosphingolipids in a chromatogram binding assay.

Solid phase radioimmunoassay and a chromatogram binding assay were used to characterize the binding specificities of five monoclonal antibodies generated from mice immunized with human tumor cell lines when tested against various glycolipids. Four antibodies derived from mice immunized with pancreatic carcinoma cells detected specifically the human blood group B determinant, Gal alpha 1 leads to 3Gal (2 comes from 1 alpha Fuc). These antibodies preferred type 2 (Gal beta 1 leads to 4GlcNAc) glycolipids. No reactivity was detected with a rat B determinant based on GalNAc. An antibody derived following immunization with a rectal carcinoma cell line was shown to have binding properties identical with those of an antibody that reacts specifically with the stage-specific embryonic mouse antigen (SSEA-1), bearing the determinant Gal beta 1 leads to 4GlcNAc (3 comes from 1 alpha Fuc) (Gooi, H. C., Feizi, T., Kapadia, A., Knowles, B. B., Solter, D., and Evans, M. J. (1981) Nature 292, 156-158). Thin layer chromatography was used to detect the binding of a monoclonal anti-tumor antibody recently shown to react with a sialylated Lea glycolipid (Magnani, J. L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H., and Ginsburg, V. (1982) Fed. Proc 41, 898) and an anti-Leb antibody (Brockhaus, M., Magnani, J. L., Blaszczyk, M., Steplewski, Z., Koprowski, H., Karlsson, K.-A., Larson, G., and Ginsburg, V. (1981) J. Biol. Chem. 256, 13223-13225) to mixtures of glycolipids from normal and tumor tissues.

Lewis blood group fucolipids and their isomers from human and canine intestine.

Glycolipids containing fucose linked to N-acetylglucosamine were isolated and characterized from 14 individual human and 13 individual dog intestines. From 8 of the dog intestines, Lewis a isomer fucolipids were isolated, all identical and having the structure Gal(beta 1 leads to 4)[Fuc alpha 1 leads to 3]GlcNAc(beta 1 leads to 3)Gal(beta 1 leads to 4)Glc-ceramide. Lewis b isomer fucolipids were isolated from 12 of the intestines, all identical and having the structure Fuc(alpha 1 leads to 2)Gal(beta 1 leads to 4)[Fuc alpha 1 leads to 3]GlcNAc(beta 1 leads to 3)Gal(beta 1 leads to 4)Glc-ceramide. Lewis a-active glycolipids were isolated as the sole major fucolipid in 6 of the human intestines and differed from the canine isomer only in the position of the linkage of galactose to N-acetylglucosamine, having the beta 1 leads to 3 (type 1) rather than the beta 1 leads to 4 (type 2) linkage. Lewis b-active fucolipids were isolated from 8 human intestines and differed from their canine isomer only in that they, too, had the type 1 rather than the type 2 oligosaccharide chain. Lewis a and b glycolipid isomers commonly co-existed in canine intestine as major fucolipids whereas Lewis a and b glycolipids did not so co-exist in human intestine. In all of the fucolipids, only hydroxylated fatty acids were present and phytosphingosine and sphingosine were the predominant long chain bases. These findings are of interest in the biosynthesis of these substances and in their genetic expression.

Blood group A antigen in human erythrocytic membranes and membrane fractions.

Erythrocytic membranes from blood group A individuals were assayed for A antigen using a quantitative hemagglutination inhibition technique. The membranes were then extracted for lipid and glycoprotein. Although some A antigen was usually found in the glycoprotein fraction, most of the activity was in the lipid fraction. The sum of A antigen activity in the lipid, glycoprotein, and membrane residue fractions only occasionally was equal to the A activity in the erythrocytic ghosts. However, when certain lipid preparations with little or no A antigen (enhancement factors) were added to the glycolipid fractions, the amount of A antigen demonstrated was usually greatly increased. Under these conditions, the sum of the fractions often was much greater than the A antigen demonstrated in erythrocytic membranes. This suggests that the organization or arrangement of A antigenic determinants in the red cell membrane may not always permit a stoichiometric reaction with anti-A molecules.

Characterization of dog small intestinal fucolipids with human blood group A activity. Differences in dog and human A-active fucolipids.

Glycolipids containing fucose (fucolipids) which carried human blood group A activity were isolated from a number of dog small intestines and analyzed. On the basis of sugar analysis, methylation, periodate oxidation, enzyme degradation, mass spectrometry, and immunologic studies, a structure is proposed for these substances. The ceramides of the dog fucolipids containing only hydroxylated fatty acids with 85% saturated and 15% monoenoic acids ranging from 16 to 25 carbon atoms. Sphingosine and phytosphingosine comprised 48% each of the long chain bases. An A-active fraction isolated from human small intestine was shown to have two components, one of which was immunologically distinct and the other identical with the dog intestinal fucolipids. The human fraction differed from the dog fucolipids in migration on thin-layer chromatography and contained two types of amino sugar substitution. It is proposed that the human fraction was composed of two fucolipids with incomplete structures.

Characterization of a human intestinal fucolipid with blood group Lea activity.

A fucolipid that carried human blood group Lea activity was isolated from human small intestine. It contianed fucose, galactose, N-acetyl glucosamine, glucose, and ceramide in a molar ratio of 1:2:1:1:1. After periodate oxidation only 1 molecule of galactose and the N-acetylglucosamine remained. Permethylation of the lipid gave derivatives of a terminal fucose and galactose residue together with 2,4,6-tri-O-methylgalactose and 2,3,6-tri-O-methylglucose. After removal of fucose the lipid could be converted to a ceramide trihexoside with beta-galactosidase, and this, in turn, to ceramide lactoside by the action of beta-N-acetylhexosaminidase. Both enzymes converted the defucosylated derivative to a ceramide monohexoside. The methylated and the methylated and reduced derivatives of the intact lipid gave ions in mass spectrometry for a terminal hexose and deoxyhexose, a terminal trisaccharide of hexose, deoxyhexose and N-acetylhexosamine, and terminal tetra-and pentasaccharides. Ceramide fragments characteristic of hydroxy fatty acids with 16, 22, 23, and 24 carbons were found together with those of phytospingosine as the major long chain base. On the basis of these results and the immunologic activity of the fucolipid, the following structure is proposed: betaGal (1 leads to 3)betaGlcNAc (1 leads to 3)betaGal (1 leads to 4)Glc-ceramide alphaFuc (1 leads to 4).

Characterization of dog small intestinal fucolipids with human blood group H activity.

Fucolipids with human blood group H activity were isolated from several dog small intestines. On the basis of mass spectrometry, periodate oxidation, enzyme degradation, methylation, and immunologic studies the following structure is proposed: Fucalpha(1 yields2)Galbeta(1 yields 4)Glc-NAcbeta(1 yields 3)Galbeta(1 yields 4)Glc-ceramide. The ceramide was shown by mass spectrometry to contain hydroxyhexadecanoic acid and phytosphingosine as major consitutuents.

Identification of a novel hepraglycosylceramide with two fucose residues and a terminal hexosamine.

A polar fucose-containing glycosphingolipid fraction isolated from dog small intestine has been characterized by mass spectrometry of intact methylated, and methylated and reduced (LiAlH4) glycolipid. The native fraction, which was homogenous on thin-layer chromatography, was shown after methylation to be a mixture of two compounds. One was identified as a hexaglycoslyceramide with the following composition and sequence: fucose-hexose(fucose)-hexosamine-hexose-hexose-ceramide, with a terminal saccharide structure similar to blood group Leb determinants. The second compound was a novel heptaglycosyceramide with the sequence: hexosamine(fucose)-hexose-tfucose)-hexosamine-hexose-hexose-ceramide. This glycolipid was also detected in human small intestine and pancreas. The dog intestinal fraction had phytosphingosine as its major base and contained almost exclusively 2-hydroxy fatty acids (16 : 0--24 : 0). The fraction of human pancreas differed in having spingosine as its major base and normal fatty acids (16 : 0--24 :0) as major acids.

Characterization by mass spectrometry of blood group A active glycolipids from human and dog small intestins.

Glycolipids with blood group A activity isolated from human and dog small intestine have been characterized by mass spectrometry of intact lipid in methylated and in methylated and reduced (LiAiH4) form. Without degradative studies the glycolipids were conclusively shown to be hexaglycosyleramides with phytosphingosine as the major long-chain base and hydroxypalmitic acid as the major fatty acid. The exact sugar ratio was hexose-hexosamine-deoxyhexose 3:2:1 and the sequence established as hexosamine-[deoxyhexose-]hexose-hexosamine-hexose-hexose-ceramide. Evidence is presented that mass spectrometry can differential between type ) and type 2 saccharide chains.

Main structures of the Forssman glycolipid hapten and a Leb-like glycolipid of dog small intestine, as revealed by mass spectrometry. Difference in ceramide structure related to tissue localization.

Two glycolipids of dog small intestine, one with Forssman activity and one with Leb-like activity, have been characterized by mass spectrometry of methylated, and methylated and reduced (LiAlH4) derivatives. The Forssman glycolipid was conclusively shown to be a pentaglycosylceramide with the carbohydrate sequence hexosamine-hexosamine-hexose-hexose-hexose-ceramide, and with sphingosine (dihydroxy base) as major long-chain base and normal fatty acids as the only fatty acids. The Leb-like glycolipid was a hexaglycosyl-ceramide with sequence fucose-hexose-[fucose-] hexosamine-hexose-hexose-ceramide and with phytosphingosine (trihydroxy base) as major long-chain base and only 2-hydroxy fatty acids as fatty acids. The difference of two hydroxy groups in the ceramide between the two glycolipids may be related to a different tissue localization. As shown by immunofluorescense study the Forssman activity was associated with the lamina propria and the Leb-like activity to the glandular epithelium of dog small intestine.