A1 Erythrocytes Research Articles

Recognition factors of Dolichos biflorus agglutinin (DBA) and their accommodation sites.

Dolichos biflorus agglutinin (DBA) is one of the well known plant lectins that are widely used in clinical serology to differentiate human blood group A1 and A2 erythrocytes and also applied to glycobiology. However, the knowledge of recognition factors of polyvalent (super) glycotopes in glycans and the roles of functional group and epimer in monosaccharide (sub-monosaccharide recognition factor) have not been well established. The size and shape of the recognition (combining) site of DBA has not been clearly defined.In this study, many importnat recognition factors of DBA-glycan binding were characterized by our established enzyme-linked lectinosorbent (ELLSA) and inhibition assays.The results of these assays showed that the intensity profile of the recognition factors for the major combining site of DBA was expressed by Mass relative potency (Mass R.P.) and shown by decreasing order of high density of polyvalent GalNAcα1 → (super glycotopes, 3.7 × 103) >> the corresponding β anomers >> monomeric GalNAcα1 → related glycotopes (GalNAc as 1.0) >> their GalNAc β-anomers >> Gal (absence of NHCH3CO at carbon-2 of GAlNAc) and GlcNAc (different epimer of Carbon-4 in GalNAc). From the all data available, it is proposed that the combining site of DBA should consist of a small cavity shape as major site and most complementary to monomeric GalNAcα → located at both terminal reducing end (Tn) and nonreducing end of glycan chains, and with a wide and broad area as subsite to accomodate from mono- to tetra-saccharides (GalNAcβ, Galβ1 → 3/4GlcNAc, lFuc1 → 2Galβ1 → 3/4GlcNAc, GalNAcβ1 → 3Galα1 → 4Galβ1 → 4Glc) at the nonreducing side. In this study, it has provided the most (comprehensive) recognition knowledge of DBA-glycan interactions at the factors of glycotope, super glycotope/sub-monosaccharide levels. Thus, it should expand and upgrade the conventional concept of the combining (recognition) site of DBA since 1980s.

A1 Erythrocytes Express Protein Structures with Blood Group A Antigenic Determinants that are not Detected on A2 Erythrocytes

A1 Erythrocytes Express Protein Structures with Blood Group A Antigenic Determinants that are not Detected on A2 Erythrocytes

Blood group antigen A type 3 expression is a favorable prognostic factor in advanced NSCLC

ObjectivesSeveral blood group-related carbohydrate antigens are prognosis-relevant markers of tumor tissues. A type 3 (repetitive A) is a blood group antigen specific for A1 erythrocytes. Its potential expression in tumor tissues has so far not been examined. Material and methodsWe have evaluated its expression in normal lung and in lung cancer using a novel antibody (A69-A/E8). For comparison an anti-A antibody specific to A types 1 and 2 was used, because its expression on lung cancer tissue has been previously reported to be of prognostic relevance. Resected tissue samples of 398 NSCLC patients were analyzed in immunohistochemistry using tissue microarrays. Results and conclusionsExpression of A type 3 was not observed in non-malignant lung tissues. A type 3 was expressed on tumor cells of around half of NSCLC patients of blood group A1 (p<0.001). Whereas no prognostic effect for A type 1/2 antigen was observed (p=0.562), the expression of A type 3 by tumor cells indicated a highly significant favorable prognosis among advanced NSCLC patients (p=0.011) and in NSCLC patients with lymphatic spread (p=0.014). Univariate prognostic results were confirmed in a Cox proportional hazards model. In this study we present for the first time prognostic data for A type 3 antigen expression in lung cancer patients. Prospective studies should be performed to confirm the prognostic value of A type 3 expression for an improved risk stratification in NSCLC patients.

Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for Organ Transplantation

Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I-IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I-IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II-based ABH antigens were expressed; type III/IV structures were not detected. Type II-based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1 , A2 , A1 B, A2 B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un-glycosylated with the terminal "A" sugar α-GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO-incompatible transplantation when interpreting anti-ABO antibodies measured by hemagglutination assays with reagent erythrocytes.

Structural modification of H histo-blood group antigen.

ABO blood group polymorphisms of humans and other primates are determined by the expression of A, B, or H antigens, which are terminal neutral glycan sequences that are abundant in glycoproteins and glycolipids. H substance is a precursor which, under the influence of genes A and B, is converted into blood group substance A or B, respectively. Given that the inactive O allele does not alter H substance, the presence of unchanged H determinants is characteristic of blood group O individuals. ABH blood group antigens in human erythrocyte membranes occur as glycolipids and glycoproteins. Among blood group ABH glycolipids on erythrocytes, substances with type 2 chains are predominant. Glycolipids with type 3 and 4 chains are present only in insignificant amounts and type 1 chain glycolipids are adsorbed from the plasma because they are not endogenous erythrocyte substances. Blood group H active antigens have been found on O-linked and N-linked carbohydrate chains1–3. The H-specific oligosaccharide unit has been identified as the trisaccharide Fucα1-2Galβ1-3GalNAc-. In the majority of these H chains, N-acetylgalactosamine carries an α2-6 neuraminic acid residue, but this structure does not seem to have any serological activity4. The blood group H determinant in human erythrocytes is carried mainly by a lacto-series type 2 chain5. Type 3 chain glycolipids have been found only in group A erythrocytes. Type 3 substance is characteristic of group A1 erythrocytes and is present in only trace amounts in A2 cells. Precursor H type 3 occurs in greater quantities in A2 erythrocytes than in A1 erythrocytes, but is absent in O and B cells. As early as the mid-20th century, studies showed that some micro-organisms have enzymatic activities that can modify ABO type6. However, it was not until the 1980s that the pioneering work of Goldstein and Lenny (New York Blood Center, NY, USA) discovered technology for enzymatic conversion of A and B antigens which could be used in blood transfusion7. The general name used for the technological concept is “Enzyme-Converted group O Red Blood Cells” (ECO-RBC). Based on the ECO-RBC concept, glycosidase and glycosyltransferase were used to modify the structure of blood group H antigens. α-1,2-L-fucosidase is a highly specific exoglycosidase that catalyses the hydrolysis of α1-2 linked L-fucopyranosyl residues from oligosaccharides, whereas plasma-derived N-acetylgalactosaminyltransferases (A-transferase) transfer N-acetylgalactosamine residues onto galactose residues in H determinants in the presence of UDP-N-acetylgalactosamine. In this study, we used α-1,2-L-fucosidase to modify the structure of type 2 chain H. The modified antigens did not show type 2 chain H antigen activity. The erythrocytes treated by fucosidase are called FM-erythrocytes. We used the A-transferase from A1 plasma to modify the structure of type 3 chain H antigens. The erythrocytes treated by A-transferase are called TM-erythrocytes.

Immunohistochemical detection of intra-neuronal VZV proteins in snap-frozen human ganglia is confounded by antibodies directed against blood group A1-associated antigens

Varicella-zoster virus (VZV) causes chickenpox, establishes latency in trigeminal (TG) and dorsal root ganglia (DRG), and can lead to herpes zoster upon reactivation. The VZV proteome expressed during latency remains ill-defined, and previous studies have shown discordant data on the spectrum and expression pattern of VZV proteins and transcripts in latently infected human ganglia. Recently, Zerboni and colleagues have provided new insight into this discrepancy (Zerboni et al. in J Virol 86:578-583, 2012). They showed that VZV-specific ascites-derived monoclonal antibody (mAb) preparations contain endogenous antibodies directed against blood group A1 proteins, resulting in false-positive intra-neuronal VZV staining in formalin-fixed human DRG. The aim of the present study was to confirm and extend this phenomenon to snap-frozen TG (n=30) and DRG (n=9) specimens of blood group genotyped donors (n=30). The number of immunohistochemically stained neurons was higher with mAb directed to immediate early protein 62 (IE62) compared with IE63. The IE63 mAb-positive neurons always co-stained for IE62 but not vice versa. The mAb staining was confined to distinct large intra-neuronal vacuoles and restricted to A1(POS) donors. Anti-VZV mAb staining in neurons, but not in VZV-infected cell monolayers, was obliterated after mAb adsorption against blood group A1 erythrocytes. The data presented demonstrate that neuronal VZV protein expression detected by ascites-derived mAb in snap-frozen TG and DRG of blood group A1(POS) donors can be misinterpreted due to the presence of endogenous antibodies directed against blood group A1-associated antigens present in ascites-derived VZV-specific mAb preparations.

Heart transplantation across antibodies against human leukocyte antigens and blood group A1 antigen. Post‐transplant follow‐up of donor reactive antibodies

We have successfully performed heart transplantation despite the most unfavourable risk factors for graft and patient survival: the presence of a high level of antibodies (Abs) against the donor's human leukocyte antigens (HLA) class I/II and blood group A1 antigens. The present study concerns post-transplant follow-up and characterization of donor reactive antibodies (DRA). Pre-transplant treatment consisted of mycophenolate mofetil (MMF), prednisolone, tacrolimus, intravenous immunoglobulin (IVIG), rituximab, protein-A immunoadsorption (PAIA) and per-operative plasma exchange. A standard triple-drug immunosuppressive protocol was used post-operatively. Abs were analyzed by the complement dependent cytotoxicity (CDC) test against donor and panel B/T cells and by flow cytometry (FlowPRA tests detecting isolated HLA class I/II antigens). Abs against the donor's erythrocytes were analyzed using a standard direct agglutination test for immunoglobulin M (IgM) Abs and a Bio-Rad AHG gel card test detecting IgG Abs and C3d. Pre-transplant treatment reduced Ab titers against the donor's lymphocytes from 128 to 16 and against the donor's blood group A1 antigen from 256 to 0. The patient was emergently transplanted with a heart from a blood group incompatible donor (A1 secretor to O). No hyperacute rejection was seen. DRA were present against all mismatched HLA class I and class II antigens at the time of transplantation; two of these DRA Abs disappeared within the first year post-transplant (anti-B62 and anti-DR4), one showed weakened reactivity (anti-A24) and one is still strongly reactive (anti-DQ3). The donor-specific CDC cross-match is still positive (titers 2 to 8). The level of panel reactive antibodies (PRA) remained unchanged from 6 months on post-transplant. Rising anti-A1 blood group Abs preceded the second rejection and were adsorbed by two blood group specific immunoadsorptions (Glycosorb)-ABO) and remained at a low level. IgM anti-A1 blood group Abs disappeared at 1 yr post-transplant and IgG Abs are still reactive with blood group A1 erythrocytes but at low titers (1 to 2). The patient is clinically well 2 years after heart transplantation despite the constant persistence of donor reactive IgG Abs against blood group A1 and HLA-DQ antigens. The reactivity of DRA against other mismatched HLA antigens disappeared or weakened during the follow-up period.

Production of a new anti-A monoclonal reagent

Monoclonal antibodies are essential tools in molecular and cellular immunology research. They have essentially replaced the polyclonal antibodies in identifying blood groups and detecting cell markers and pathogenic agents. The aim of the present study is to produce monoclonal antibody identifying the ABO blood groups using the murine hybridoma technology. An anti-A monoclonal antibody A907 was selected and estimated for its use in the manufacture of a reagent anti-A. The selected antibody specifically reacts with A1, A2, A1B and A2B erythrocytes. It does not recognize B, O, A3 and Ax erythrocytes. The A907 monoclonal antibody can be used in blood grouping in association with a reagent recognizing the A weak phenotypes.

Production of a new anti-A monoclonal reagent

Monoclonal antibodies are essential tools in molecular and cellular immunology research. They have essentially replaced the polyclonal antibodies in identifying blood groups and detecting cell markers and pathogenic agents. The aim of the present study is to produce monoclonal antibody identifying the ABO blood groups using the murine hybridoma technology. An anti-A monoclonal antibody A907 was selected and estimated for its use in the manufacture of a reagent anti-A. The selected antibody specifically reacts with A1, A2, A1B and A2B erythrocytes. It does not recognize B, O, A3 and Ax erythrocytes. The A907 monoclonal antibody can be used in blood grouping in association with a reagent recognizing the A weak phenotypes.

Enhanced invasion of blood group A1 erythrocytes by Plasmodium falciparum

Invasion of the malaria parasite Plasmodium falciparum into human erythrocytes is a highly complex multi-step procedure. It involves parasite ligands on the merozoite interacting with receptors on the erythrocyte surface. P. falciparum is able to exploit multiple receptor ligand interactions to achieve invasion and to date, several erythrocyte surface receptors have already been identified [1]. However, several as yet unidentified receptors on the erythrocyte surface have also been postulated to play a role in invasion and the specific molecular interactions between merozoite surface ligands and erythrocyte receptors remain elusive. Inhibition of RBC invasion would prevent infection and consequently disease, therefore considerable effort has gone into identification and characterization of molecules involved in this process.

Studies on Blood-Groups AEnzymes.1 and A2

Blood-group O and A2 erythrocytes were treated with the A1-gene-dependent and A2-gene-dependent N-acetylgalactosaminyl transferases in the present of UDP-N-acetyl[14C]galactosamine. Although the transfer of N-acetyl[14C]galactosamine with A2 transferase was slower than with A1 enzyme, group O as well as A2 cells became agglutinable by anti-A1 reagents when incubated with both transferases. Fractionation of the labelled erythrocyte stroma into glycoprotein and glycolipid components showed an approximately equal distribution pattern of radioactivity in all experiments. Likewise, when the short-chain glycolipids and polyglycosylceramides isolated from the labelled stroma were further analyzed by thin-layer chromatography, no major differences were detected in the chromatographic profiles of O and A2 cells when treated with either transferase. These observations indicate that (a) the blood-group-H-type oligosaccharide chains of A2 cells may be similar to those of group-O cells and (b) the serological differences between A1 and A2 cells are likely to be due to a lower density of A-antigenic sites on A2 cells.

ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin Ulex europaeus agglutinin I

The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA1) (10 micrograms/ml) was found to increase the rate constant of Cl- efflux into 100 mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mM K+ oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of Cl- efflux by 20-50%. The acceleration of Cl- efflux by UEA1 was completely blocked by 10 microM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl- efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na+ pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein.

Role of ABH blood group antigens in the stimulation of a DIDS-sensitive Ca2+ influx pathway in human erythrocytes by Ulex europaeus agglutinin I and a monoclonal anti A1 antibody

Of eleven agglutinating lectins tested, only one, Ulex europaeus agglutinin I (UEA1), stimulated Ca2+ uptake in quin2-loaded erythrocytes by about 2-fold. UEA1 is known to be an α-l-focuse and ABH blood group specific lectin. The 45Ca2+ influx induced by UEA1 was absent in the presence of fucose (5 and 15 mM) and depended on the ABH blood group of the donor, the stimulatory potency of the lectin decreasing in the order H > A2 > A1. Ca2+ entry blockers, such as cobalt and verapamil, did not affect the 45Ca2+ influx induced by UEA1. 4,4′-Diisothiocyanatostilbene-2, 2′-disulfonic acid (DIDS) inhibited dose-dependently with a Ki of 1–2 μM. 10 μM DIDS, 10 μM 4,4′-dinitristilbene-2,2′-disulfonic acid (DNSD) and 20 μM dipyridamole fully blocked the 45Ca2+ influx induced by UEA1. The effect of UEA1 on 45Ca2+ influx was absent in K+ and Mg2+ media and was less pronounced in choline than in Na+ media. The 45Ca2+ influx induced by the lectin was abolished by preincubation with 12-O-tetradecanoylphorbol 13-acetate (TPA, 60 ng/ml). A monoclonal antibody raised against A1 erythrocytes (Bric 54) accelerated 45Ca2+ influx in quin2 loaded A1 erythrocytes b by about 2-fold. No effect was seen in A2 and H erythrocytes. The 45Ca2+ influx elicited by Bric 54 exhibited a sensitivity towards inhibition by DIDS and TPA, as well as a dependence on the cation composition of the incubation medium similar to that observed with UEA1. The effects of UEA1 and Bric 54 were not additive. These observations suggest that the Ca2+ influx induced by UEA1 and Bric 54 is mediated by the same transport pathway. Since both the lectin and the antibody exhibit ABH blood group specificity, it appears reasonable to conclude that ABH antigens can serve as recognition sites for activation of a Ca2+ influx pathway in human erythrocytes, which is sensitive to inhibitors of the band 3 anion-exchanger.

A study of immunogold-labelled blood group A erythrocytes in the scanning electron microscope.

This report presents binding patterns on A1, A2, A3, Ax and Ae1 erythrocytes of a monoclonal anti-A (A 003) antibody which reacts predominantly with difucosylated A oligosaccharides, visualized with colloidal gold particles in the backscattered electron imaging mode of a scanning electron microscope. A relatively weak labelling was found on most A1 cells, while the labelling in subgroups A2, A3 and especially Ax appeared relatively stronger. Very few Ae1 cells were labelled. The results emphasize the qualitative uniqueness of A1 and suggest that many Ax cells have high proportions of difucosylated A oligosaccharides. Labelling variations were found between different A3 and Ax traits, and in all subgroups between cells and even between different parts of the cells. No immunolabelling differences were found in relation to secretor status.

Detection of ABO blood group-active glycolipids extracted from red cell membrane and heat hematoma using TLC-immunostaining.

Glycolipids extracted from groups A, B, and O erythrocytes were developed on thin-layer plates; their ABO blood group antigenicities were detected by immunostaining method using avidin-biotin-complex (ABC). Among series of glycolipids of different flow rates, antigen-specific staining was observed in five bands from group A1 erythrocytes, four bands from group B, and two bands from group O. Monoclonal anti-A, -B, and -H antibodies specifically stained glycolipids from A1, B, and O erythrocytes, respectively. ABO blood grouping was possible from 5 g of epidural heat hematoma of a charred body by this method. ABC immunostaining on thin-layer chromatography is a useful and reliable method for ABO blood grouping in forensic practice.

STUDIES ON MOUSE MONOCLONAL ANTI-A AND ANTI-A, B ANTIBODIES OBTAINED BY IMMUNIZATION WITH BLOOD GROUP A SECRETOR SALIVA

Three monoclonal anti-A (3H9, 5C6, 4D8) and one monoclonal anti-A, B (2D10) antibodies were obtained from a mouse by immunization with blood group A secretor saliva. The agglutination of erythrocytes of A subgroups by 3H9 was similar to that by human anti-A serum, and 3H9 was directed to the type 2 chain A structure. 5C6 agglutinated A1 erythrocytes and reacted especially with difucosyl type 2 chain A and type 3 chain A structures. Antibody 4D8 showed stronger agglutination of A2 erythrocytes than the others, and reacted with both type 3 chain and type 4 chain A structures. By using these antibodies, the determination of A subgroups and characterization of conformational structures of the A determinants are possible. Antibody 5C6 was inhibited rather strongly by A non-secretor saliva. Thus, this antibody is useful for application to test specimens of legal medicine, such as examination of blood or salivary stains for A antigens.The reactivity of these antibodies with erythrocyte membrane glycoproteins was analyzed by immunostaining after SDS-polyacrylamide gel electrophoresis. 3H9 reacted with band 3 and band 4.5, 4D8 with glycophorins, and 5C6 did not react with glycoproteins. These results suggest that blood group A determinants are present in erythrocyte membrane glycoproteins and that their sugar chain structures are different depending on the glycoproteins, polylactosaminoglycan type 2 chain A in band 3 and band 4.5 but GalNAcα1→3 (Fucα1→ 2) Galβ1→3GalNAc in glycophorins.

Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances.

This is the first report of nucleotide and translated amino acid sequences of the variable region light (VL) and heavy (VH) chains of mouse monoclonal hybridoma anti-blood group A and B substances, the combining sites of which have been mapped. Monoclonal hybridoma anti-A and anti-B produced in BALB/c mice by immunization with A or B blood group substances, with A1 erythrocytes, and water-soluble blood group A substance or with synthetic B determinants coupled to bovine serum albumin or to O erythrocytes have been characterized immunochemically. To relate the immunochemical properties of the monoclonals to their primary structures, we have cloned and sequenced cDNAs of variable regions of light and heavy chains of two anti-A and two anti-B. The anti-A hybridomas have very similar combining site specificities and have almost identical VH sequences belonging to the J558 germ-line family, but their VL are from different germ-line VK gene families. The two anti-B hybridomas have different combining site specificities and use the same VL which differs completely from the anti-A VL; their VH are derived from different VH germ-line genes belonging to the J606 family. The results suggest that the heavy chains play a major role in determining the specificities of the antibody combining sites, with only minor contribution of VL. Additional sequence data on monoclonal antibodies of defined specificity for blood group substances are needed for further insights into the genetic and structural basis for their specificities.

Murine Monoclonal Antibodies to Human A Erythrocytes: Differential Reactivity with N‐acetyl‐D‐galactosamine

Six different hybridoma cell lines were obtained producing IgM monoclonal antibodies (MAbs) against human blood group A antigen. All these MAbs agglutinate the A1, A1B and A2 erythrocytes, while only one of them agglutinates the A2B erythrocytes. The agglutination activity of 3 of these MAbs is inhibited by N-acetyl-D-galactosamine (D-GalNAc). Dissociation constants of complexes of these MAbs with D-GalNAc are approximately 2.2 X 10(-3) mol/l.

Monoclonal antibodies specific for type 3 and type 4 chain-based blood group determinants: Relationship to the A1 and A2 subgroups

Two monoclonal antibodies, specific for A type 3 and A type 4 blood group determinants, are described. These antibodies recognized A1 but not A2 erythrocytes. A third monoclonal antibody showing specificity for A type 3 and A type 4, and also for H type 3 and H type 4, did not discriminate between A1 and A2 erythrocytes. On red cells these three antibodies recognized glycosphingolipids and binding to glycoproteins could not be demonstrated. On paraffin-embedded tissue sections the three antibodies labelled a supranuclear area, characteristic of the Golgi apparatus, of all cells producing A antigens. This labelling occurred irrespective of the A1, A2 status.

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.