Biochemical and enzymatic characterization of blood group ABH and related histo-blood group glycosphingolipids in the epithelial cells of porcine small intestine

Non-acid glycosphingolipids were isolated from small intestinalepithelial cells of a single blood group A pig. One very predominantblood group compound was obtained chemically pure upon HPLCfractionation. It was characterized by mass spectrometry and¹H NMR spectroscopy to be the type 1 chain blood group A hexaglycosylceramide.Support for the presence of minute amounts of additional A glycolipidswas obtained by mass spectrometry and immunostaining of TLCplates with anti-A antibodies specific for A type 2 chain, Atype 3 and 4 chain, and the ALe^b determinant. Among precursorchains, globoside (type 4) and lactotetraosylceramide (type1) were immunologically identified, whereas no neolactotetraosylceramide(type 2) and gangliotetraosylceramide reactivities were detected.We addressed the question whether the predominant expressionof type 1 chain based A glycolipids reflects a restricted glycolipidprecursor chain specificity of the     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

Non-acid glycosphingolipid expression in plasma of an A1 Le(a-b+) secretor human individual: identification of an ALeb heptaglycosylceramide as major blood group component.

Total non-acid glycosphingolipids were isolated from plasma of an A1 Le(a-b+) secretor individual with Refsum's disease (phytanic acid storage disease). The glycolipids were separated into 11 fractions by open column chromatography and by HPLC. The fractions were analyzed by thin-layer chromatography and tested for different blood group A activities as well as blood group Le(a )and Leb activity. The fractions were structurally characterized by proton NMR spectroscopy and FAB mass spectrometry and in selected cases by EI mass spectrometry of the permethylated and permethylated-reduced derivatives. Degradation analysis was performed on partially permethylated or permethylated-reduced alditol acetates. The dominating blood group compound was found to be a blood group A active type 1 chain difucosylheptaglycosylceramide. Other blood group compounds were identified as a blood group A active type 1 chain monofucosylhexaglycosylceramide, a blood group Leb hexaglycosylceramide, a blood group H active type 1 chain pentaglycosylceramide, and a globotetraosylceramide (the P-antigen). The presence of a Le(a) glycosphingolipid and blood group A type 3/4 chain structures were also found by immunostaining. Glucosyl-, lactosyl-, and globotriaosylceramides were the dominating short chain compounds. The amount of phytanic acid incorporated into the monoglycosylceramide fraction was found to be less than 5% of the fatty acids.     

Tissue specificity of glycosphingolipids as expressed in pancreas and small intestine of blood group A and B human individuals

A chemical investigation has been done on blood group active glycosphingolipids of both small intestine and pancreas from two individuals, one blood group A and one blood group B. Total non-acid glycolipid fractions were prepared and the major blood group fucolipids present were purified and structurally characterized by mass spectrometry, proton NMR spectroscopy, and degradation methods. The glycolipid structures identified were a blood group Leb hexaglycosylceramide, a B-hexaglycosylceramide with a type 1 (Gal beta 1 leads to 3GlcNAc) carbohydrate chain, A-hexaglycosylceramides with types 1 and 2 (Gal beta 1 leads to 4GlcNAc) carbohydrate chains, a B-heptaglycosylceramide with a type 1 carbohydrate chain, and A-heptaglycosylceramides with type 1 and 2 carbohydrate chains. In addition several minor glycolipids having more than seven sugar residues were detected by thin-layer chromatography. The small intestine and pancreas had some distinct differences in their expression of the major fucolipids. The small intestine contained only glycolipids based upon type 1 carbohydrate chain while the pancreas had both type 1 and type 2 structures. The intestines contained mainly difucosyl compounds while the pancreas tissues contained both mono- and difucosyl glycolipids. Monofucosylglycolipids based on both types 1 and 2 saccharides were present in one pancreas while the other one contained only monofucosylcomponents based on type 1 chain. The ceramides of the intestinal glycolipids were found to be more hydroxylated (trihydroxy long-chain base, hydroxy fatty acids) compared to the pancreas glycolipids (dihydroxy long-chain base, non-hydroxy fatty acids).     

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies.

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.     

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A₂ kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A₂ kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Galα1–4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of Gaα1–3(Fucα1–2)Galβ-HexNac-Galα1–4Galβ-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant glood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which migh explain the low expression of type-4 chain blood group heptaglycosylceramides in human blood group B kidneys.     

Glycosphingolipid expression in pig aorta: identification of possible target antigens for human natural antibodies

Total non-acid glycosphingolipids were isolated from the aortas of more than 80 pigs. The glycolipids were separated by HPLC, analysed by thin- layer chromatography, and tested for reactivity with monoclonal anti- blood group antibodies. The fractions were structurally characterized by NMR spectroscopy and mass spectrometry. Reactivity with both anti- blood group A and H antibodies was seen. The major glycosphingolipid constituents were globotri- and globotetraosylceramides and blood group H pentaglycosylceramides based on type 1 and type 2 core saccharide chains. Globopentaosylceramides, blood group H hexaglycosylceramides based on type 4 chain, and blood group A hexaglycosylceramides based on type 1 core chain were also present. Two structures, that may be important targets for human antibodies initiating hyperacute rejection following pig to human xenotransplantation, were present as minor constituents compared to the blood group components. These were Galalpha1,3neolactotetraosylceramide and a Galalpha1, 3Lexstructure. A Leb/Y hexaglycosylceramide was also present.      

Structural characterization of non-acid glycosphingolipids in kidneys of single blood group O and A pigs

Total non-acid glycosphingolipids were isolated from the kidneys of single pigs serologically typed on their red blood cells as blood groups O and A. Glycolipid species were purified by HPLC and structurally characterized by thin-layer chromatography, mass spectrometry, proton NMR spectroscopy, degradation analysis, and reactivity with various monoclonal antibodies, Gal alpha 1-4Gal-specific E. coli bacteria, and lectins. Glucosyl-, globotriaosyl-, and globotetraosylceramides were the predominant molecular species with lactosyl- and globopentaosylceramides (IV3GalGb4Cer) as abundant constituents too. Small amounts of galactosyl- and digalactosylceramides were also present. In the blood group O pig kidneys, blood group H antigens based on four different core saccharides (types 1, 2, 4, and lactosyl core) were identified and the major blood group structure was V2FucIV3Gal-Gb4Cer. In the kidneys from the blood group A pig the corresponding blood group A antigens were found and in addition, a type 3 chain blood group A antigen was indicated by mass spectrometry and by its reactivity with a monoclonal antibody. Trace amounts of the type 2 chain-based X and Y antigens were found while blood group B antigens and the type 1 chain based Lewis antigens could not be detected. The ceramide part of the glycolipids was mainly composed of dihydroxy 18:0 long chain bases and non-hydroxy 16:0-24:0 fatty cids.     

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.     

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.     

Redefinition of the Carbohydrate Binding Specificity of Helicobacter pylori BabA Adhesin

Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fucα2Galβ3(Fucα4)GlcNAc; Le(b)) and the H type 1 determinant (Fucα2Galβ3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Le(b) and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), and a mixture of three complex glycosphingolipids (Fucα2Galβ4GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fucα2Galβ3GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, and Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A.     

Human blood group glycosphingolipids of porcine erythrocytes.

Two glycosphingolipids with human blood group A and H antigenicity were isolated from porcine erythrocyte membranes which were obtained from the pooled blood. The yield of the A- and H-antigenic glycolipids was approximately 0.2 and 0.1% of total neutral glycolipids, respectively. No B antigen was detected. Through several methods the porcine erythrocyte antigens were all found to belong to lactoseries (type 1 chain), IV2Fuc alpha, IV3GalNAc alpha Lc4Cer for type A and IV2-Fuc alpha Lc4Cer for type H, in contrast to the antigenic glycolipids in human erythrocytes, which mostly belong to neolactoseries (type 2 chain). The constituent fatty acids of the A antigen were 75% normal acids and 25% 2-hydroxy acids, and the long chain base was 95% sphingenine. This is the first demonstration of the A- and H-antigenic glycolipids on erythrocytes of pig in whose gastric mucin the human blood group A and H substances have been demonstrated.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Blood group ABH-active substances.

High resolution nuclear magnetic resonance spectra of permethylated and permethylated-reduced (LiAlH₄) derivatives were recorded in chloroform solution for the following glycosphingolipids with known structure: lactotriaosylceramide, neolactotetraosylceramide (paragloboside), two blood group H-active pentaglycosylceramides (type 1 and type 2 saccharide chains, respectively), a B-active hexaglycosylceramide, an A-active hexaglycosylceramide, and an A-active octaglycosylceramide. Good quality and resolution allow a clear-cut diagnosis of α-anomeric protons of Fuc, Gal, and GalNAc, and in most cases of all β protons. Upon reduction there is a strong deshielding effect on H-1 of Gal of Galβ1 → 3GlcNAc but not on Gal of Galβ1 → 4GlcNAc. It is therefore possible to differentiate type 1 and type 2 chains by this method, a structural difference of importance for serological specificity. Nuclear magnetic resonance spectroscopy may therefore provide conclusive information on the anomeric structure of the immunodeterminant of blood group-active glycolipids using the same derivatives as for sequence analysis by mass spectrometry.     

Transient expression of type 2 chain in A-active hexaglycosylceramide of rat small intestine at weaning time. Demonstration by affinity chromatography and ceramide glycanase hydrolysis of A-active glycosphingolipids followed by gas chromatography and mass spectrometry of permethylated hexasaccharides

The small intestine of 15- to 23-day-old rats was cut into four segments from the duodenum to the ileum. Neutral glycosphingolipids were purified from each segment and submitted to thin-layer chromatography and immunostaining with the A005 monoclonal anti-A antibody. This antibody detected an hexaglycosylceramide located mainly in the duodenum during the postnatal development. In order to characterize hexaglycosylceramides, blood group A-active glycolipids were purified by affinity chromatography on immobilized Helix pomatia lectin in organic solvent. Hexaglycosylceramides (A-6) were subsequently isolated by preparative thin-layer chromatography and hydrolyzed with ceramide glycanase. The free hexasaccharides were permethylated and analyzed by gas chromatography. Two peaks were detected in varying ratios during development, corresponding to type 1 and type 2 chain A hexasaccharides. Gas chromatography clearly demonstrated that type 2 A-6 occurred in the duodenum of developing rats, and that a shift from type 2 to type 1 A-6 occurred with growing age. The change from type 2 to type 1 chain was also assessed by methylation analysis, and by the variation of the characteristic fragmentations of type 1 and type 2 chain hexasaccharides upon mass spectometry of the permethylated A-6 oligosaccharides from the duodenum of 19-day-old and adult rats.     

Characterization of a membrane-bound arginine-specific serine protease from rat intestinal mucosa

Previously we isolated and characterized a membrane-bound, arginine-specific serine protease from pig intestinal mucosa [J. Biol. Chem. 269, 32985-32991 (1994)]. For further characterization of this type of enzyme, we cloned a cDNA from rat intestinal mucosa encoding the precursor of a similar protease. The partial amino acid sequences determined for the pig enzyme were found to be shared almost completely by the rat enzyme. The serine protease domain of the rat enzyme, heterologously expressed in Escherichia coli, specifically cleaved Arg (or Lys)-X bonds with a marked preference for Arg-Arg or Arg-Lys, similar to the pig enzyme. The mRNA for the rat enzyme was shown to be distributed mainly in intestine, and the enzyme was detected in the duodenal mucosa as a 70 kDa protein. Immunohistochemical analysis of the small intestinal tissue showed that the enzyme is localized mainly on brushborder membranes.     

Incompatible A antigen expressed in tumors of blood group O individuals: immunochemical, immunohistologic, and enzymatic characterization

Monofucosyl type 1 chain A (type 1 Aa) and difucosyl type 1 chain A (ALeb), but not other types of A antigens, have been detected by application of carrier type-specific monoclonal anti-A antibodies (AH21 and HH3) in colonic tumors of blood group O individuals. An A-transferase activity (UDP-Gal-NAc:H-alpha-GalNAc transferase) was demonstrated in the extract of one of the O tumors expressing A antigen. The incidence of A antigen expression in O tumors was found to be two out of 15 cases, based on TLC immunostaining of glycolipid extracts, and five out of 50 cases, based on immunofluorescent staining of tumors with AH21 and HH3 antibodies.     

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.     

The sugar chain structures of ABO blood group active glycoproteins obtained from human erythrocyte membrane

The glycoproteins of human erythrocyte membrane have two groups of sugar chains with blood type ABH determinants, which are quite distinct in their molecular sizes. A neutral sugar chain and an acidic sugar chain, which belong to the small size group, were isolated from the glycoproteins obtained from the erythrocyte of blood type O individuals, and their structures were elucidated as Fucalpha1 leads to 2Galbeta1 leads to 3N-acetylgalactosaminitol and Fucalpha1 leads to 2Galbeta1 leads to 3(AcNeualpha2 leads to 6)N-acetylgalactosaminitol, respectively. The molecular weight of the large sugar chains with ABH determinants were estimated to be more than 4000. Both large and small neutral sugar chains of membrane glycoproteins obtained from blood type O erythrocyte could serve as acceptors of alpha-N-acetylgalactosaminyltransferases purified from milk of blood type A1 and A2 individuals, producing the same radioactive sugar chain distribution patterns. However, the acidic sugar chain with the H determinant could not serve as an acceptor of these enzymes.     

Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). II. Differential conversion of different H substrates by A1 and A2 enzymes, and type 3 chain H expression in relation to secretor status

The novel A-associated H antigen (type 3 chain H), described in the accompanying paper (Clausen, H., Levery, S.B., Kannagi, R., and Hakomori, S. (1986) J. Biol. Chem. 261, 1380-1387), as well as globo-H were found to be present in greater quantity in A2 erythrocytes than in A1 erythrocytes. A1 erythrocytes contain the repetitive A epitope (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), which is defined by A1-specific monoclonal antibody TH-1, in addition to globo-A. The ability of alpha-GalNAc transferase from A1 and A2 serum to catalyze the conversion of type 2 chain H, type 3 chain H, and globo-H to type 2 chain A, type 3 chain A, and globo-A, respectively, was compared. The conversion to type 3 chain A and globo-A occurred to a minimal degree in the presence of the A2 enzyme as compared with the A1 enzyme, particularly at low substrate concentration. Although a lower conversion from type 2 chain H to type 2 chain A was also observed in the presence of the A2 enzyme than in the presence of the A1 enzyme, the conversion of type 2 chain H to type 2 chain A was less restricted than the type 3 chain conversion catalyzed by the A2 enzyme, particularly at low substrate concentration. The conversion from globo-H to globo-A was essentially absent in the presence of the A2 enzyme. Since the expression of type 1 chain H in erythrocytes is dependent on secretor status, the distribution of type 3 chain H and globo-H in erythrocytes from secretors and non-secretors was compared. These antigens appeared to be present in the same quantity in erythrocytes of secretors and nonsecretors.     

The occurrence of the feline A blood group antigen on lymphocytes

Sera from 300 cats were tested for the presence of anti-lymphocytic antibodies. One hundred and nineteen sera showed some activity with the majority (79) reacting only with lymphocytes from blood group A cats. Absorption of two such sera with A, AB and B erythrocytes and absorption of AB system reagents with lymphocytes from A and B blood group cats demonstrated that the A antigen is expressed on both erythrocytes and lymphocytes. Blood group and lymphocyte typing tests of foetuses indicated that the A antigen is present on these tissues as early as 46 days gestation. The erythrocytic B antigen could not be demonstrated on lymphocytes although a single antiserum, which reacted against lymphocytes from group B cats, was found. Several sera containing anti-lymphocytic antibodies which were not related to the AB type were also detected.