MINIREVIEW TIGG Vol. 3 No.13 (September 1991) Structure and Possible Functional Significance of Human Red Cell Blood Group Antigens(ABH, Lewis, Ii, P-

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1 MINIREVIEW Structure and Possible Functional Significance of Human Red Cell Blood Group Antigens(ABH, Lewis, Ii, P-related, Duffy, and Antigens on Glycophorin A) May-Jean KING International Blood Group Reference Laboratory, South Western Regional Transfusion Centre, Bristol BS 10 5ND, England, FAX: Abstract Human blood group antigens on red cells can be broadly classified into carbohydrates and proteins. The former category includes ABH, Lewis(Le), Ii, and P-related antigens, and the latter category is represented in this minireview by the Duffy antigens(fyaand Fyb), and the M and N antigens carried on glycophorin A. The ABH and Ii antigens are located on the polylactosaminoglycans of glycoproteins Band 3 and Band 4.5, and on polyglycosylceramides. The Lea, Leb and the P-related antigens are associated only with glycosphingolipids. These carbohydrate-dependent antigens are expressed not only on red cells, but they are also detected on various epithelial tissues, organs, and secretions. Some of them are tumor-associated as their expressions on certain malignant tissues are incompatible with the blood groups of the cancer patients. The Duffy antigens and glycophorin A are erythroid specific. They have been identified as separate ligands to which the merozoites of Plasmodium knowlesi(or P. vivax) and Plasmodium falciparum attach respectively at the asexual stage of the life cycle of these malarial parasites. Key Words: blood group antigenslerythrocyteslglycoproteinlglycolipidlmalarial parasites A. Introduction In 1901 Landsteiner discovered that there were immunological differences among red cells of various individuals. These differences were attributed to the blood group antigens, which were expressed in accordance to the Mendelian Law of Inheritance(cited in ref.1). At present there are nineteen blood group systems, including over 200 red cell antigens(2), which can be classified broadly into carbohydrate-dependent and protein-dependent antigens. In the past 30 years, the most-studied and well-characterized blood group antigens are of carbohydrate nature, namely, ABH, Lewis(Le), Ii, P- related, and P1(see recent reviews: ref. 3, 4). These antigens are synthesized by sequential actions of glycosyltransferases that catalyze the transfer of monosaccharides from nucleotide sugars onto carbohydrate precursors of glycoprotein or glycolipid. Thus the glycosyltransferases are the primary gene products while the carbohydrate-dependent antigens are sec- 315

2 ondary products of the blood group genes (see review, ref. 5). The majority of the human blood group antigens are protein determinants carried on red cell membrane glycoproteins (see review: ref. 6). The amino acid and cdna sequences of only a small number of these membrane components have been fully characterized, for example, Glycophorins A, B, C, and D(see reviews: ref. 7 and 8). This minireview presents the chemical structures of the carbohydrate-dependent antigens(namely, ABH, Lewis, Ii, P, and P1) and brief descriptions of glycophorin A and the component that carries Duffy antigens. All these antigens have been associated with different biological functions. B. Chemical Structure and Composition of Blood Group Antigens B.1. The ABH and Lewis Antigens The ABO blood group system remains the most important in transfusion practice because of the common occurrence of anti-a and anti-b antibodies, in persons whose red cells lack the corresponding antigens(1). In group 0 individuals, whose red cells carry the H antigenic determinant Fucƒ (1-2)Ga1ƒÀ-R, both anti-a and anti-b are present. TheƒÀ-galactosyl residue in the H disaccharide forms part of the acceptor sequence at the non-reducing terminal of 4 types of carbohydrate chains(table 1). Both type 1 and type 2 chains are present on water-soluble glycoproteins, such as mucus glycopro- Table 1. Structure for antigens ABH, Lea, Leb, X and Y 316

3 teins(9,10), or on oligosaccharides from human milk(11) and urine(12). Human red cell glycosphingolipids are mainly of type 2 chain(13) while type 3 and type 4 chain sequences are present as minor components. There are two ƒ -2-L-fucosyltransferases capable of synthesizing the H determinant. The ƒ -2-L-fucosyltransferase encoded by the H gene is found in tissues of mesodermal origin, while that encoded by the secretor gene(se) is present in the tissues of epithelial origin(14,15). The A and B antigenic determinants are trisaccharides carrying terminal a(1-3)galnac and a(1-3)gal, respectively(table 1). The A- and B- gene specified glycosyltransferases have a stringent acceptor requirement in that only the ƒà-galactosyl residue substituted with ƒ (l-2) fucose (i.e. the H determinant) is the acceptor(s). Recent results from cdna sequencing show that the specificity of these two glycosyltransferases is defined by substitution of four different amino acid residues(16). The LE gene encodes an ƒ -4-L-fucosyltransferase which synthesizes the Lea determinant only on type 1 chain. The Leb determinant is difucosylated, which encompasses both the H and Lea determinants(table 1). The Lea and Leb antigens on human red cells are glycosphingolipids of plasma origin(17,18). Nevertheless, the Lea determinant was found recently on a minor red cell glycolipid consisting of a hybrid type 1 and type 2 chain sequence(19). The X antigen (Lex or SSEA-1)(Table 1) is synthesized on type 2 chain by an -3-L-fucosyltransferase(20). The Y antigen(or Let) is ƒ a difucosylated structure consisting of both X and the H antigen (Table 1). X and Y antigens are found predominantly in tissues or secretions and they are not detected on human red cells. Human red cell glycoproteins carrying ABH antigens are in greater abundance than those of glycolipids(21). These glycoproteins are the transmembrane components Band 3(the anion transport protein) and Band 4.5(the glucose transporter)(22, 23); each carries a highly branched N-glycan consisting of polylactosaminyl sequences(24, 25). On the contrary, the ABH antigens found on secretory glycoproteins are carried on oligosaccharide chains in ƒ -O-glycosidic linkage to serine or threonine in the polypeptide backbone(9). The ABH glycolipids from red cells have carbohydrate chains varying from 8 residues to over 30 sugar units per molecule (26). The latter type is designated polyglycosylceramide, the characteristics of which are repeating lactosaminyl units and more soluble in aqueous solvent than in organic solvent becaue of the large number of sugar residues(27). On treating intact red cells with endo-ƒà-galactosidase, there is a marked reduction in ABH and Ii activity(28). A cryptantigen, Tk, is uncovered(29), which is detectable by agglutination with the lectins from Grif fonia sirnplicifolia II, formerly known as Bandeiraea simplicifolia I1(30), and Vicia hyrcanica(31). The important subgroups of A are Al and A2. The basic difference between red cells of these two phenotypes lies in the types of glycosphingolipid: a glycolipid with type 3 317

4 chain repetitive A epitope and globo-a with type 4 chain are present only on Al red cells(table 2)(32). In addition to structural differences in oligosaccharides, the A1- and A s-specific -3-N-acetylgalactosaminyltransferases in serum and ƒ ovarian cyst fluid exhibit different properties in respect to ph optima pi, and requirements for divalent cation and acceptors(33-35). B. 2. The i and I antigens Blood group i and I antigens are carried on the internal portion of the branched N-glycan on ABH glycoproteins or glycolipids, and on a subpopulation of gangliosides (10-16 carbohydrate residues) of human red cells(36). The i antigen is a linear chain Ga1ƒÀ(1-4)G1cNAcƒÀ(1-3)GalƒÀ(1-4)G1cNAcƒÀ(1-3)GalƒÀ(1-4)Glc-Cer. Two repeating units of Ga1ƒÀ(1-4)G1cNAc and G1cNAcƒÀ(1-3)Gal are essential for the full expression of i activity(37). Inhibition of human anti-i autoantibodies using ovarian cyst glycopeptides(38), human red cell macroglycolipids(39), and bovine gangliosides(40) revealed heterogeneous binding specificity and the importance of a branched structure GaLƒÀ(1-4)GIcNAcƒÀ(1-6)Gal (Fig, 1) for the I antigenic activity(41, 42). B. 3. The Pk, P and Pl Antigens The blood group P system initially consisted of three antigens (1*, P, P1), which are absent on the red cells of individuals with the p phenotype(43). All p individuals have anti- P and anti-pk antibodies. Approximately 50% of pregnancies in p women with enhanced anti-p or anti-pk activity have had repeated spontaneous abortions in the first trimester(44). The enhanced anti-p produced is due to a secondary immune response elicited by the pregnant woman in vivo against the placental P antigen(globoside)(45). All the blood group P- active and P-related substances of human origin are glycosphingolipids but Pl glycoprotein is found in sheep hydatid cyst fluid(46) and in pigeon ovomucoid(47, 48). The Pk and P antigens are detected on red cells, erythroblasts, platelets, megakaryocytes, fibroblasts and endothelial cells(49, 50). Chemical structures for these antigens(table 3) reveal that Pk is the precursor of P whereas the precursor for P l is paragloboside Gala(1-4)GIcNAcƒÀ(1-3)GaIƒÀ(1-4)Glc(1-1)Cer(51). Thus in the blood group terminology(2), the blood group P sys-, Table 2. Oligosaccharides associated with group Al red cells 318

5 Macroglycolipid (with HI antigenic activity) isolated from human red cells Fig, 1. Heterogeneity of the I antigenic activity tem has only the P1 antigen while a separate "collection" includes P, Pk, and a new determinant called Luke(LKE)(52), which is closely related to SSEA-4(Table 3). The structures of ABH, Ii, X, Y, and P1 suggest that their precursor in vivo is probably paragloboside. The compound specificities in some human blood group antibodies(1), e.g. anti-hi, -Hi, -AI, -P1I and -HILeb, can be explained by the close proximity of these antigens on carbohydrate chains. B. 4. Antigens on Glycophorin A Glycophorin(GP) A(Mr 37 kd and 131 amino acid residues) is a transmembrane sialoglycoprotein(sgp) with a highly glycosylated extracellular domain: 15 0-glycosylation sites(53) and one N-glycan of the complex type on ASN-26 (54). The 0-glycans are mono-, di-, and tri-sialylated derivatives of the disaccharide Ga1B(1-3)GalNAca-O-Ser/Thr (Table 4)(55). Blood group antigens M, N, and Wrb are carried on this SGP(see review, ref. 7). The polymorphism between M Table 3. Antigenic determinants for P, I, LKE and P1 319

6 and N antigens is attributed to different amino acid residues in positions 1 and 5 at the NHz-terminal(53): Amino terminus S&-Ser*-Thr*-Thr*-j-Val-Ala-Met of GP AM Amino terminus Leu1-Ser*-Thr*-Thr*-Glu-Val-Ala-Met of GP AN *: 0-glycosylation sites Inhibition of anti-m and anti-n antibodies with synthetic peptides showed that the NH2-terminal Serine and Leucine respectively are the primary determinants for M and N antigens(56). The Wrb antigen is located approximately between residues 62 to 72 on GP A(7), which is absent from those En(a-) erythrocytes, which lack GP A but there is an enhanced glycosylation of Band 3(57, 58). Tn, T and Cad are carbohydrate-dependent antigens associated with the type of 0-glycans found predominantly on glycophorin A. Tn syndrome is an acquired disorder characterized by defective galactosylation of GalNAcƒ -O-Ser(Thr) (59) chains on hemopoietic cells due to a deficiency of membrane-bound ƒà(l-3) galactosyltransferase(60). Although GalNAcƒ -O-Ser(Thr) is the Tn determinant and the predominant structure on Tn red cells, sialosyl-tn(61) and varying amounts of sialylated oligosaccharides(table 4) are also detected(62). The T determinant GalƒÀ(1-3)Ga1NAc is the core structure of the oligosaccharides shown in Table 4. It is exposed after desialylation in vivo and in vitro by the action of neuraminidases which are released from organisms such as Vibrio cholerae, Clostridium perfringens, pneumococci, and influenza viruses. In patients that have these bacterial infections, there is a transient pseudo autoimmune "hemolytic anemia", because the desialized red cells are agglutinable by the anti-t present in the serum of the patients(see review: ref. 63). Cad is an inherited condition and three phenotypes have been described(cad 1, 2, and 3)(64). The Cad determinant is Table 4. Proposed structures of 0-linked oligosaccharides isolated from glycophorin A 320

7 a pentasaccharide, Ga1NAca(1-4)[NeuAcƒ (2-3)]GalƒÀ(1-3)[NeuAca(2-6)]Ga1NAc-ol, which has been isolated from glycophorin A (65) and as a ganglioside (Ga1NAcB-sialosylparagloboside)(66). A serologically related antigen called Sdm has not yet been isolated from human red cells. However, Sdm-active oligosaccharide has been isolated from the Tamm-Horsfall urinary glycoprotein in 0-linked and N-linked oligosaccharide chains. Its structure is Ga1NAcƒÀ(1-4) [NeuAcƒ (2-3)] Ga1ƒÀ(1-4)G1cNAcƒÀ(1-3)Gal-R(67). B. S. The Dully Antigens The Duffy blood group system is polymorphic and six antigens are defined by reaction with antibodies: Fy, Fyb, Fy3, Fy4, Fy5, and Fy6(64, 68). The Duffy null genotype Fy(a-b-) is common amongst American Blacks and West Africans. Red cells from these individuals are resistant to infection in vivo by Plasmodium vivax and in vitro by Plasmodium knowlesi(69). Biochemical information is available only on the membrane component that carries Fya antigen. It has a M r of 35 kd-55 kd(70, 71), which was reduced to 261W after endo-f treatment of intact red cells(72). This glycoprotein aggregates readily upon isolation from red cells. Peptide fingerprint analysis detected minor differences between Fyaand Fyb antigens(73). C. Functional Aspects of the Selected Blood Groups C.1. Differentiation or Maturation Markers for Normal Hemopoietic Cells and Tissue The bone marrow is primarily responsible for erythrocyte, neutrophil, and platelet production. Transition from fetal to adult erythropoiesis is virtually complete months after birth. During this period of red cell differentiation, the most notable change in blood group activity is the conversion of the weak I and strong i activities into strong I and weak i which are often associated with normal adult red cells(74). The result is a significant increase in the amount of lactosaminoglycan and its branching on mature red cells(75). A higher than normal level of i antigen on red cells from an adult indicates premature release of red cells from bone marrow; a condition usually found in patients who are affected by anemia(64). The influence of the secretor gene in the expression of ABH, X and Y antigens on human hemopoietic cells and epithelial tissues have been reviewed by Oriol and coworkers (7, 76). Although the LE (Lewis) gene and SE gene are inherited independently, the Lewis phenotype in body fluids and tissues is influenced by the secretor status. ABH and Leb antigens are absent on glycoproteins in secretions or epithelial tissues from an individual whose genotype is sese. An understanding of this relationship is important when studying aberrant blood group antigen expression in carcinomas of gastrointestinal tracts(see section C.2). Neutrophils and granulocytes carry X antigen(lacto-n-fucopentaose V or CD15) on the polylactosaminyl structures of glycoprotein and glycolipid (77, 78). The CD15 antigen mediates cellular adhesion of 321

8 activated platelets to neutrophils and monocytes(79). The sialyl Lex is the ligand recognized by endothelial leukocyte adhesion molecule-1(elam-1)(80), which is a member of the LEC-CAM or selectin family of adhesion molecules. On platelets the ABH antigens with type 1 chain and Lea glycosphingolipids are absorbed from plasma while those expressed on type 2 chain are intrinsic(81). The P antigen can be another marker of red cell differentiation because globoside is not detected on other blood cells such as erythroblast precursors, proerythroblasts, granulocytes, monocytes and most peripheral blood lymphocytes(50). Glycophorin A is erythroid specific and these molecules are expressed on the surface membrane of the earliest morphologically recognizable erythroid cells, proerythroblasts(82). 0-glycosylation of GP A increases with red cell maturation while the M and N blood group antigens are expressed at a late stage( the polychromatic stage) of red cell differentiation (83). The actual function of this major SGP is still not known as individuals totally lacking GP A on their red cells[e. g. En(a-) or MkMk phenotype] show no clinical symptoms nor is the life span or physiological function of the red cells affected. However, the GP A locus may be susceptible to somatic mutation as the variant frequencies of the hemizygotes for M and N allele and homozygote for M allele(mm) among the Atomic bomb survivors increased with radiation dose(84). C. 2. Blood Group Antigens as Tumour Markers Most of the changes observed in the glycosylation of malignant tissues are due to incomplete carbohydrate chain synthesis, a de novo appearance of incompatible antigens on tumors, or a change in SE gene controlled expression of antigens (see reviews: Ref. 85, 86). Such changes are illustrated by gastric carcinomas: some show a lack of ABH antigens with a high level of I activity(87) whereas in others there is a marked expression of Lea, instead of Leb and sialylated Lee, in secretor individuals(88). A recent study on malignant bladder urothelium showed that the deletion of the ABH blood group antigens on tumors was due to an absence of the activity of the A- and B-gene specified glycosyltransferases while theƒ -4-Lfucosyltransferase activity was the same in normal and malignant tissues(89). On the other hand, patients with ALL and CML had reduced levels of H-specific fucosyltransferase activity, which returned to normal levels during clinical remission (see review, ref. 90). The expression of Tn antigen on red cells was reported in some cases to be associated with hemolytic anemia, leukemia and preleukemic states(91-93). Springer first reported the detection of T and Tn antigens on primary and metastatic tumors, e, g, breast carcinoma(see review, ref. 94). However, some workers found that the T antigen was not necessarily associated with malignancy because this antigen was also detected on ductal and lobular cells of normal breast tissue and a broad spectrum of non-malignant pathological conditions of the mammary gland(95, 96). Nevertheless, at a histological 322

9 level, T-antigen might be useful in the identification of premalignant changes(97). Only the Tn antigen has consistently been proven to be of predictive value on the invasiveness of breast cancer(98, 99). Recently sialosyl-tn is an additional cancer-associated antigen for human colon cancer(100). C. 3. Interaction with Microbes C. 3. a. Ligands for Malarial Parasites Human malaria is caused mainly by Plasmodium falciparum or Plasmodium vivax when the merozoite form of these parasites invade red cells to begin the asexual phase of the life cycle. Invasion starts with attachment of merozoites, followed by junction formation between the apical zone of the parasite and the red cell, and invagination of the red cell(see review: ref. 101). Using red cells carrying rare blood group phenotypes in in vitro culture has enabled the identification of different membrane components acting as ligands in the interaction with 2 different species of parasites. The surface receptor on P, falciparum was fast found to interact with glycophorin A, irrespective of the MN status of the red cells. Both En(a-) and Wrb red cells in culture resisted invasion when compared with normal red cells(102,103). Resistance to parasite invasion could be conferred onto normal red cells by treating them with neuraminidase or trypsin. An addition of al-acid glycoprotein restored the invasion of these enzyme-treated red cells(104). These findings indicated that GP A and sialic acid residues were both required by the parasite. When the Thai-2 parasites were found to be able to grow in Tn red cells as well as in normal red cells, this suggested involvement of two different receptors for sialic-acid independent and sialic acid dependent ligand, respectively (105). A recent study showed that there is a switch mechanism within Pm falciparum to enable the parasite to adapt to red cells which are deficient in sialic acid(106). P. knowlesi, a simian equivalent of the human parasite P, viva:, requires entirely different red cell ligands to that of P. falciparum for invasion. The Duffy blood group antigens (Fya and Fy b) and band 3 are believed to be involved(see review: Ref. 101). Two separate stages have been identified: attachment and internalization of parasite. The recognition and apical re-orientation of merozoites is identical for both Duffypositive and Duffy-negative red cells. However, the parasites tend to detach from Daffy-negative red cells without gaining entry into the red cells. This effect indicates that the Duffy determinants are required by the parasites to complete the second stage of invasion (69). C. 3. b. Ligands for Bacterial Adhesion Glycosphingolipids, more often than glycoproteins, act as receptors for bacterial adhesion to tissue surfaces prior to infection. Three types of carbohydrate sequences are recognized by several groups of bacteria: internal lactosylceramide, GalNACƒÀ(1-4)Gal in the asialogangliosides, and Gala(1-4)Gal in the globoseries(see review: Ref. 107). The Gala(1-4)Gal sequence is shared with the blood group P and P,

10 antigens. Three strains of E.coli show specific interaction with different blood group antigens. The ONAP strain binds to both the group A determinant and the Gala(1-4)Gal sequence in Globo A type 4 chain (Table 2)(108). The strain (with M-fimbriae) reacts with the amino terminal of GP AM octapeptide and 0-glycosidic oligosaccharides(109). E.coli with Pap-2-encoded fimbriae recognizes SSEA-4 on human red cells and Bowman's capsules in human kidney (110). The effect following infection with Mycoplasma pneumoniae is a transient autoimmune disor4er due to the production of anti- I autoantibodies. The ligand recognized by this organism is (2-3) sialosyl N-acetyllactosamine on the polylactosaminoglycans of red cells(1 l 1). D, Concluding Remarks The clinical importance of any particular blood group system is determined by two factors: the ability of the antibodies to cause in vivo red cell destruction, and the frequency that these antibodies are found amongst the population. The biological functions of blood group antigens on human red cells per se are not clear. Polymorphisms in blood group antigens do not offer individuals any evolutionary advantage or disadvantage. The exception is the situation where individuals of Duffy null genotype are not infected by P. vivax. At present, this is the only example for the specific involvement of a blood group antigen in the well-being of an individual. The contribution of the ABH, Ii, Lewis, X, Y and P antigens may lie in their wide tissue distribution where these structures serve as differentiation or maturation markers for normal tissues, and possibly the binding sites for microbes in bacterial infections. Expression of appropriate blood group antigens in tissues may be essential for cell-cell contact. In malignancy, there is de-differentiation as shown by changes in glycosyltransferase activities and the patterns of glycosylation. It is important to establish the mechanisms that regulate the synthesis of blood group specified glycosyltransferases at the molecular level. Acknowledgments The author thanks the Mitsubishi Kasei Institute of Life Sciences(Tokyo) for the financial support provided during her visit to the Laboratory of Glycoconjugates Research in July Thanks are also due to Dr. M. Reid and Ms. J. Poole of the International Blood Group Reference Laboratory(Bristol) for their comments, and Dr. M. Uchikawa of the Japanese Red Cross(Tokyo) for the translation of this minireview. ƒ Reference 1. Mollison, P. L. (1979) Blood Transfusion in Clinical Medicine. 6th Edition. Blackwell Scientific Publications 2. Lewis, M., and 32 members of the ISBT Working Party on Terminology for Red Cell Surface Antigens (1990) Vox Sang. 58, Oriol, R., Le Pendu, J., and Mollicone, R. (1986) Vox Sang. 51, Clausen, H., and Hakomori, S. (1989) Vox Sang. 56, Watkins, W. M., Greenwell, P., Yates, A. D., and Johnson, P. H. (1988) Biochimie 70,

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