Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). I. Isolation and chemical characterization

A series of blood group H antigens reacting with monoclonal antibody MBrl has been found in human blood group A and AB erythrocytes, but not in O or B erythrocytes. These H antigens are clearly different from the globo-H structure (Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer), which was previously isolated from O erythrocytes and is also reactive with the MBrl antibody. The new series of H antigens associated with blood group A has been characterized as having TLC mobilities which approximately coincide with those of H2, H3, and H4 glycolipids. One of these A-associated H antigens, having a similar TLC mobility as the H2 glycolipid, was isolated from A erythrocytes and was characterized by 1H NMR spectroscopy, methylation analysis, and enzymatic degradation as having the structure shown below: (formula, see text). The structure represents a precursor of the repetitive A epitope attached to type 2 chain, previously called type 3 chain A (Clausen, H., Levery, S. B., Nudelman, E., Tsuchiya, S., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1199-1203). This A-associated H structure is hereby called type 3 chain H.     

Glycosphingolipids of porcine blood: human blood group A and H antigens with type 1 chain in erythrocytes and plasma

Glycosphingolipids were purified from porcine erythrocytes and plasma. Two minor glycolipids with human blood group A and H antigenicities were found in both sources as components. The two antigenic glycolipids were identified as a hexaglycosylceramide (IV3 alpha GalNAc,IV2 alpha Fuc-Lc4Cer) for the A antigen and pentaglycosylceramide (IV2 alpha Fuc-Lc4Cer) for the H antigen and belonged to lactoseries (type 1 sugar chain) in contrast to those with neolacto core (type 2 sugar chain) in human erythrocytes, thereby endorsing biochemically the previous serological observations that the A antigen on porcine erythrocytes is uptake from plasma, probably the H antigen being the case. In addition to major glycolipids of globoseries in red cells and plasma, a variety of acidic glycolipids including two classes of sulphatides (sulphated galactosylceramide and sulphated lactosylceramide) and five classes of gangliosides (GM3, GD3, GM1, fucosyl GM1 and GD1a) containing N-acetylneuraminic acid and N-glycolylneuraminic acid were obtained from plasma.     

Characterization of blood group ABO(H)-active gangliosides in type AB erythrocytes and structural analysis of type A-active ganglioside variants in type A human erythrocytes

Several monosialogangliosides containing the type A-active epitope have been detected in type A erythrocytes on immunological analysis with a monoclonal antibody, and three of them were purified by repeated silica bead column chromatography and by scraping from the TLC plate. Two of these A-active gangliosides were characterized by methylation analysis by GC/MS, negative SIMS, MALDI-TOF/MS, proton nuclear magnetic resonance spectroscopy, and immunological assays, and their structures were concluded to be as follows. A-active ganglioside I:A-active ganglioside II:The reactivity of the purified gangliosides to the anti-A monoclonal antibodies (mAbs) exhibited enhancement after removal of the sialic acid. Therefore, the sialic residue has been shown to inhibit the binding to the terminal A-active epitope through the formation of an immune complex. To confirm the presence of A- (including S-A-I, -II and -III) and B-active gangliosides, the reactivity of anti-A and -B mAbs were investigated using total gangliosides from type A, -B and -AB erythrocytes on TLC plate. The results were that the gangliosides from types A and AB showed positive reaction to anti-A mAbs, whereas in the anti-B mAbs binding the gangliosides from types B and AB were positive. Thus, it revealed that A-active gangliosides were present in type A and -AB, and B-active gangliosides in types B and AB. As there was no difference in respective gangliosides on type AB erythrocytes of 22 individuals, both A- and B-active gangliosides are equally present in type AB erythrocytes. The biological significance of these A- and B-active ganglioside variants remains vague at present. As these molecules exhibit different reactivities to the anti-A mAbs, it is very likely that they can regulate the antigenicity of the A-epitope on the cell surface.     

Differential recognition of the type I and II H antigen acceptors by the human ABO(H) blood group A and B glycosyltransferases

The human ABO(H) blood group A and B antigens are generated by the homologous glycosyltransferases A (GTA) and B (GTB), which add the monosaccharides GalNAc and Gal, respectively, to the cell-surface H antigens. In the first comprehensive structural study of the recognition by a glycosyltransferase of a panel of substrates corresponding to acceptor fragments, 14 high resolution crystal structures of GTA and GTB have been determined in the presence of oligosaccharides corresponding to different segments of the type I (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->3)-beta-D-GlcNAcp-OR, where R is a glycoprotein or glycolipid in natural acceptors) and type II (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->4)-beta-d-GlcNAcp-OR) H antigen trisaccharides. GTA and GTB differ in only four "critical" amino acid residues (Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268). As these enzymes both utilize the H antigen acceptors, the four critical residues had been thought to be involved strictly in donor recognition; however, we now report that acceptor binding and subsequent transfer are significantly influenced by two of these residues: Gly/Ser-235 and Leu/Met-266. Furthermore, these structures show that acceptor recognition is dominated by the central Gal residue despite the fact that the L-Fuc residue is required for efficient catalysis and give direct insight into the design of model inhibitors for GTA and GTB.     

Further characterization of type 2 and type 3 chain blood group A glycosphingolipids from human erythrocyte membranes

Blood group A glycosphingolipids with slow chromatographic mobilities have been separated systematically with an improved chromatographic procedure, and their structures have been analyzed by application of a panel of monoclonal antibodies defining A determinants carried by type 1, type 2, type 3, and type 4 carbohydrate chains as well as by 1H NMR spectroscopy and methylation analysis. Of several A-active fractions, previously termed Aa, Ab, Ac, and Ad, in decreasing order of thin-layer chromatographic mobility, the third fraction (Ac) was characterized as containing one type 3 chain A component and one type 2 chain A component without branching, which have been termed type 3 chain Ab and nor-Ac, respectively. (Formula: see text). The major component present in the fourth A-active fraction (Ad) was isolated and characterized as a branched type 2 chain glycolipid formerly termed Ac. The major component in the fifth A-active fraction (Ae) was identified as a branched type 2 chain A previously termed Ad. The structures of Ac (n = 1) and Ad (n = 2) are (Formula: see text).     

Molecular modeling of glycosyltransferases involved in the biosynthesis of blood group A,blood group B,Forssman, and iGb3 antigens and their interaction with substrates

A terminal alpha1-3 linked Gal or GalNAc sugar residue is the common structure found in several oligosaccharide antigens, such as blood groups A and B, the xeno-antigen, the Forssman antigen, and the isogloboside 3 (iGb3) glycolipid. The enzymes involved in the addition of this residue display strong amino acid sequence similarities, suggesting a common fold. From a recently solved crystal structure of the bovine alpha3-galactosyltransferase complexed with UDP, homology modeling methods were used to build the four other enzymes of this family in their locked conformation. Nucleotide-sugars, the Mn2+ ion, and oligosaccharide acceptors were docked in the models. Nine different amino acid regions are involved in the substrate binding sites. After geometry optimization of the complexes and analysis of the predicted structures, the basis of the specificities can be rationalized. In the nucleotide-sugar binding site, the specificity between Gal or GalNAc transferase activity is due to the relative size of two clue amino acids. In the acceptor site, the presence of up to three tryptophan residues define the complexity of the oligosaccharide that can be specifically recognized. The modeling study helps in rationalizing the crystallographic data obtained in this family and provides insights on the basis of substrate and donor recognition.     

Blood group A glycolipid antigen expression in kidney, ureter, kidney artery, and kidney vein from a blood group A1Le(a-b+) human individual. Evidence for a novel blood group A heptaglycosylceramide based on a type 3 carbohydrate chain

Kidney, ureter, kidney artery, and kidney vein tissue were obtained from a single human transplant specimen. The donors erythrocyte blood group phenotype was A1Le(a-b+). Total non-acid glycolipid fractions were isolated and individual glycolipid components were identified by immunostaining thin layer plates with a panel of monoclonal antibodies and by mass spectrometry of the permethylated and permethylated-reduced total glycolipid fractions. The dominating glycolipids in all tissues were mono- to tetraglycosylceramides. In the kidney, ureter, and artery tissue less than 1% of the glycolipids were of blood group type, having more than 4 sugar residues. In contrast, 14% of the vein glycolipids were of blood group type, and the dominating components were type 1 chain blood group H pentaglycosylceramides and A hexaglycosylceramides. Trace amounts of structurally different blood group A glycolipids (type 1 to 4 core saccharide chains) with up to 10 sugar residues were found in the kidney, ureter, and vein tissues, including evidence for a novel blood group A heptaglycosylceramide based on the type 3 chain in the vein. The only detected A glycolipid antigen in the artery tissue was the blood group A difucosyl type 1 chain heptaglycosylceramide (ALeb) structure. Blood group Lewis and related antigens (Lea, Leb, and ALeb) were expressed in the kidney, ureter, and artery, but were completely lacking in the vein, indicating that the Le gene-coded alpha 1-4-fucosyltransferase was not expressed in this tissue. The X and Y antigens (type 2 chain isomers of the Lea and Leb antigens) were detected only in the kidney tissue.     

Presence of H type 3/4 chains of ABO histo-blood group system in serous cells of human submandibular gland and regulation of their expression by the secretor gene (FUT2).

We have investigated by immunochemistry the distribution of H Type 3/4 chains of the ABO histo-blood group system in human submandibular gland using a monoclonal anti-H MBr1 antibody specific for H Type 3/4 chains, and have found the expression of H Type 3/4 chains was mainly in the serous cells. Serous cells from secretors were stained by MBr1 but not by anti-A and anti-B antibodies, whereas serous cells from nonsecretors exhibited a negative reaction with MBr1. Mucous cells were not stained by MBr1. Only a few striated duct cells showed a weak reaction with anti-H MBr1. These results suggested that the H Type 3/4 chains were distributed predominantly in the serous cells of the human submandibular gland and that secretor Type alpha(1,2)fucosyltransferase (Se enzyme) controlled the synthesis of H Type 3/4 chains in vivo. Saliva also contained H Type 3/4 chains, which were controlled by the secretor gene (FUT2). The differences in the distributions of H Type 1, H Type 2, and H Type 3/4 chains of the ABO histo blood group system in the submandibular gland are discussed.     

Absorption of anti-blood group A antibodies on P-selectin glycoprotein ligand-1/immunoglobulin chimeras carrying blood group A determinants: core saccharide chain specificity of the Se and H gene encoded alpha1,2 fucosyltransferases in different host cells

To specifically eliminate recipient anti-blood group ABO antibodies prior to ABO-incompatible organ or bone marrow transplantation, an efficient absorber of ABO antibodies has been developed in which blood group determinants may be carried at high density and by different core saccharide chains on a mucin-type protein backbone. The absorber was made by transfecting different host cells with cDNAs encoding a P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) chimera (PSGL-1/mIgG(2b)), the H- or Se-gene encoded alpha1,2-fucosyltransferases (FUT1 or FUT2) and the blood group A gene encoded alpha1,3 N-acetylgalactosaminyltransferase (alpha1,3 GalNAcT). Western blot analysis of affinity-purified recombinant PSGL-1/mIgG(2b) revealed that different precursor chains were produced in 293T, COS-7m6, and Chinese hamster ovary (CHO)-K1 host cells coexpressing FUT1 or FUT2. FUT1 directed expression of H type 2 structures mainly, whereas FUT2 preferentially made H type 3 structures. None of the host cells expressing either FUT1 or FUT2 supported expression of H type 1 structures. Furthermore, the highest A epitope density was on PSGL-1/mIgG2(2b) made in CHO-K1 cells coexpressing FUT2 and the alpha1,3 GalNAcT. This PSGL-1/mIgG(2b) was used for absorption of anti-blood group A antibodies in human blood group O serum. At least 80 times less A trisaccharides on PSGL-1/mIgG(2b) in comparison to A trisaccharides covalently linked to macroporous glass beads were needed for the same level of antibody absorption. In conclusion, PSGL-1/mIgG(2b), if substituted with A epitopes, was shown to be an efficient absorber of anti-blood group A antibodies and a suitable model protein for studies on protein glycosylation.