C O N T E N T S
The ABH antigens are molecules on red blood cells, and in secretions of secretors, that define the blood groups A, B, AB and O.
The ABH antigens are not primary gene products but instead they are the enzymatic reaction products of enzymes called glycosyltransferases?. The ABO system occurs as a result of polymorphism of complex carbohydrate structrures of glycoproteins and glycolipids? expressed at the surface of erythocytes or other cells, or present in secretions, as glycan units of mucin glycoproteins. Immuno-dominant structures of A and B antigens, GalNAc alpha1->3 (Fuc alpha1->2) Gal- and Gal alpha1->3 (Fuc alpha1->2) Gal-, respectively, are synthesized by a series of reactions; the A and B transferases encoded by the functional alleles (A and B alleles) of a single gene at the ABO locus, catalyze the last step of the synthesis, while the transferase coded by the O allele is non-functional; therefore, the acceptor substrate, (H antigen: Fuc alpha1->2 Gal-) remains without a further modification and the A and B determinants are absent.
Our current knowledge of the inmmunochemistry of the ABH and Lewis antigens is mainly the result of several decades of systematic investigation by two laboratories, that of Morgan and Watkins at the Lister Institute in England, and Kabat's group at Columbia University College of Physicians and Surgeons in the United States. These studies were performed on water-soluble substances isolated from secretions, primarily from ovarian-cyst fluid.
The ABH substances purified from secretions are glycoproteins composed of approximately 80 per cent carbohydrate and 20 per cent amino acids. They are heterodisperse, with an average molecular weight of 300,000, but the molecules of different sizes have similar composition and serological properties. The glycoproteins contain a peptide backbone to which multiple oligosaccharide chains are attached through an alkali-labile glycosidic bond to the hydroxyl group of serine or threonine. Most of the oligosaccharide chains are linked to the backbone through an N-acetylgalactosamine? residue. The carbohydrate moiety of the ABH and Lewis glycoproteins consists primarily of four sugars, D-galactose?, L-fucose?, N-acetylgalactosamine? and N-acetylglucosamine. A small amount of N-acetylneuraminic acid ([sialic acid]?), generally less than 1% but occasionally as much as 18 % is found in many cysts. This sugar does not appear to have blood group specificity and in high concentrations may interfere with the expression of blood group activity. The amino acid compositions of the different blood group glycoproteins are similar to each other, and unrelated to blood-group specificity. Serine and threonine constitute more than 40 per cent of the total amino acids, and proline and alanine are also present in relatively large amounts, but aromatic and sulfur containing amino acids are virtually absent.
The ABH and [Lewis Blood Group? Lewis glycoproteins] possess a common basic structure, and their blood-group specificity is determined by the sequence and linkage of sugars at the terminal nonreducing end of the carbohydrate chains. The number of chains bearing antigenic determinants has been estimated at 40 to 100 per 300,000 molecular weight. (14,15) The structures of these antigenic determinants, which have recently been elucidated by hapten-inhibition studies.
There are two types of backbone structures, Type I chains, which contain galactose linked -(1-3) to N-acetylglucosamine, and Type 2 chains in which the linkage is -(1-4). Oligosaccharides with these nonreducing terminal ends do not possess blood-group specificity, but can be detected immunologically by their cross-reaction with horse antiserum prepared against Type 14 pneumococcal polysaccharide. A glycoprotein with oligosaccharide chains terminating with either of these sequences has been termed a “precursor substance," and a substance of this type has been isolated from ovarian-cyst fluid. The presence of fucose on C-2 of the terminal galactose of either Type 1 or Type 2 chain produces an H determinant. The presence of fucose linked to C-4 of N-acetylglucosamine in a Type I chain results in Lea activity, but a Type 2 oligosaccharide containing fucose linked to C-3 of N-acetylglucosamine has very weak Lea activity. The simultaneous presence of both fucose substituents on a Type I chain results in the appearance of a new antigenic specificity, Leb , and loss of most of the H and Lea activities. Type 2 difucosyl chains have weak Leb activity.
The A determinant consists of the Type 1 or 2 H structure plus a terminal nonreducing N-acetylgalactosamine-linked alpha(1-3) to galactose. Similarly, the B determinant consists of the H structure plus a terminal alpha(1-3) galactose residue. H activity is lost when the additional sugars are added. The Type 2 Group A determinant containing two fucose residues has much less A activity than the monofucosyl Type 2 determinant shown in Figure 1, possibly because of conformational changes produced in the nonreducing end of the determinant. Isolation of a difucosyl Type I Group A determinant has not been reported. The unbranched trisaccharide
alpha (1->3) beta (1->3)
(GalNAc ------------aGal -----------aGNAc)
has appreciable A activity but it is less active than the fucose containing tetrasaccharide. It is not clear whether the fucose residue makes contact with the antibody binding site or stabilizes a particular conformation of the terminal trisaccharide but is not itself in contact with the antibody.
The structures of most of the ABH and Lewis determinants have been established, but the complete structure of the glycoproteins remains to be ascertained. There are at least four types of oligosaccharide chains that have blood-group specificity, Type 1 and 2, monofucosyl and difucosyl chains (as noted above in the Group A determinant), and very short chains without blood-group activity may also exist. The various types of oligosaccharide chain may not be distributed equally among the glycoprotein molecules synthesized by a single individual. Moreover, Lloyd et al recently isolated a branched oligosaccharide containing both Type 1 and Type 2 chains, and they proposed the existence of "megalosaccharide" chains that contain two determinants.
Most glycoproteins isolated from a single cyst have multiple specificities. For example, an A1 glycoprotein usually has strong A1 and Leb properties, and weak H and Lea activity. The evidence available indicates that most of these diverse specificities are present on each molecule, rather than segregated on different molecules. This was shown by precipitation of a blood-group substance with an antibody specific for a single determinant, and the demonstration that the other specificities were removed from the supernatant. Since each oligosaccharide depicted in Figure 1 has only one strong haptenic activity, it is likely that the multiple specificities result from different chains attached to a common backbone. In the example noted above, the Leb and H activities would result from the presence of incomplete chains that lack the terminal N-acetylgalactosamine. In addition, some antibodies may be able to react with an internal determinant in a completed chain.
ABO genes consist of at least 7 exons, and the coding sequence in the seven coding exons spans over 18kb of genomic DNA. The single nucleotide deletion, found in a large number (but not all) of O alleles and responsible for the loss of the activity of the enzyme, is located in exon 6. The first of the seven nucleotide substitutions which distinguish the A and B transferases, resides in coding exon 6; exon 7, the largest of all, contains the other six nucleotide substitutions which result in four amino acid substitutions that differentiate the A and B transferases. Among those, substitutions responsible for alterations at two sites (residues 266 and 268) determine the A or B specificity of the enzyme (Yamamoto and Hakamori). In addition to four common alleles (A1, A2, B and O), numerous alleles which encode glycosyltransferases with changes in activity and/or specificity have been identified. Function of proteins
The primary gene products of functional alleles are glycosyltransferases. The A alleles encode UDP-GalNAc: Fuc alpha1->2 Gal alpha1->3 N-acetyl-D-galactosaminyltransferase (alpha 1->3 GalNAc transferase or histo-blood group A transferase). The B alleles encode UDP-Gal: Fuc alpha1->2 Gal alpha 1->3 galactosyltransferase (alpha 1->3 galactosyltransferase or histo-blood group B transferase). O alleles encode proteins without glycosyltransferase function. The function of ABH antigens remains unknown.
Gene expression is not restricted to erythroid tissues but occurs universally in most epithelial and endothelial cells. Expression of the antigens may undergo changes during development, differentiation and maturation. Aberrant expression is often observed in human pre-malignant and malignant cells.
It was known early in the century that ABH substances occur in human tissues and secretions in two forms, water-soluble and alcohol-soluble, and that persons with these substances in saliva (secretors) have more water-soluble substances in their tissues than those lacking the substance in their saliva (nonsecretors). These studies have been extended and refined in recent years by the use of sensitive mixed agglutination and immunoflourescence technics that allow precise localization of the substances to individual cells. Immunofluorescent staining has revealed ABH substances in the cell membranes of all vascular endothelial cells, and certain epithelial cells. 1 The membrane antigens are alcohol soluble and occur in secretors and non-secretors? alike. The positive epithelia are stratified or pseudostratified, and include the skin, tongue, esophagus, lower genitourinary tract and uterine cervix.
ABH substances are secreted by [Mucin? mucous glands] in many organs, including the upper respiratory tract, the gastrointestinal tract from the esophagus through the colon and the uterine cervix. (1) The synthesis of blood-group substances in superficial glands of the gastric and small-intestine mucosa is regulated by the [19q13.3 secretor gene]?. Large amounts of ABH material are found all secretors, but abundant Leb substance and no ABH substance is seen in non-secretors. Glands situated more deeply in the mucosa of the pylorus and small intestine (Brunner's glands) produce A and B substances without regard to secretor status. These substances are not extracted by fixation of the tissues with alcohol and are probably glycoproteins. The gastric parietal glands also produce A and B substances in both secretors and nonsecretors, but this material is alcohol soluble and is probably glycosphingolipid in nature.(2,3,4,5,6,7)
Expression of ABO antigens has been shown by immunohistochemical techniques on the hepatic artery, portal vein, capillary, sinusoidal lining cells, and bile duct epithelium but not on the bile ductule or hepatocytes (8)
The prostate glands and the lactating mammary glands of secretors also produce ABH substances.(9) The pattern of secretion by the breast is unusual in that abundant H substance is produced by secretors of all ABH phenotypes, but much less A substance, and virtually no B substance, is detectable in the breast or in milk. (10) Synthesis of ABH substances in the exocrine acini of the pancreas and the secretory cells, of sweat glands is not regulated by the secretor gene. ABH substances are detectable in the plasma of non-secretors? and secretors; the latter tend to have higher [Isohemagglutinin Titer? titers], but there is considerable overlap between the two groups. (11,12)
Blood Group A and B glycoproteins have been isolated from urine by King, (13) and Lundblad and Berggard. (14) These materials are similar to ovarian cyst glycoproteins in their chemical composition, but the preparation of Lundblad contained glucose, which has not been detected in other human blood group glycoproteins. The glucose could be a constituent of a glycoprotein contaminant since no evidence was presented that the glucose-containing material was precipitable by specific antiserum. In another study, Lundblad (15) isolated pentasaccharides with A and B activity from urine. Both types of oligosaccharide? contained one glucose residue on the reducing end, and the A compound had in addition one mole each of [GalNAc? N-acetylgalactosamine] and galactose?, and two moles of fucose?. The origin of these materials is unknown. Blood-group substances have been detected in the collecting tubules and calyxes of secretors, but it is not clear whether they are synthesized by the kidney or merely excreted. The membranes of epithelial cells of the lower urinary tract contained alcohol-soluble ABH antigens, presumably glycosphingolipids?, and it has been suggested that the urinary oligosaccharides may be derived from a cell-membrane substance. 16
Szulman (17,18) has studied the appearance of ABH substances in cell membranes and secretions of human embryos. The membrane substances are found first in the epithelium all vascular endothelium of the youngest embryos examined, estimated at about five weeks of gestational age. At this time all epithelia contain the ABH substances except those of the nervous system, adrenal glands and liver. The [Antigen? antigens] disappear from these epithelia in an orderly and predictable manner as evidences of morphologic differentiation appear, and by about the end of the third intrauterine month, the-adult pattern of distribution is achieved. ABH substances appear in secretions at eight to nine weeks' ovulation age in the salivary glands and stomach, and then appear throughout the gastrointestinal tract and other characteristic locations.
These antigens are found in secretions throughout the body and on the surface of epithelial and endothelial cells.
As will become apparent in the list of alleles, subgroups of each category of ABO alleles (A, B or O) have been documented, first serologically, as they exhibited unique phenotypes under defined conditions; also by characteristic transferase activities, and more recently, at the nucleotide sequence level. The latter studies are revealing that DNA variation (single or multiple mutations,rearrangements) occur in each category, often in a recurrent fashion, i.e. in a number of alleles, identical mutations occur at one or more identical sites. The incidence of the occurrence of the various alleles in world populations is not known (except for selected populations, such as the Japanese, see Ogasawara K. et al.) and there seems to be a difference in frequency among different ethnic groups. In the Table below, percent allelic frequency for each allele represents the sum of all the alleles in that category.
When referring to the list of alleles, allele A101 is taken as reference. For intronic sequences described in Seltsam et al. or Roubinet et al. nucleotide positions are numbered starting from the first nucleotide of each intron. Results, for the same allele, originating from different laboratories (as different regions of the allele may have been sequenced) are referred to when they are not identical; exonic mutations are similarly referred to for the origin of their documentation.
Among South American Indians, whereas the incidence of A1, A2 or B allele is rare, that of the O allele is 90-100%. The distribution of ABO genotypes including allele frequencies in a sample of Chinese or white European population was recently documented in an article 19
1. Hearn, V. M., Smith, Z. G., and Watkins, W. M. a-N-Acetyl-Dgalactosaminyltransferase associated with human blood-group A character. Biochein. J. 109:315-317, 1968
2. Szulman, A. E. Chemistry, distribution, and function of blood group substances. Ann. Rev. Med. 17:307-322, 1966
3. Glynn, L. E., Holborow, E. J., and Johnson, G. D. Distribution of blood group substances in human gastric and duodenal mucosa. Lancet 2:1083-1088, 1957
4. Glynn, L. E., and Holborow, E. J. Distribution of blood-group substances in human tissue. Brit. M. Bull. 15:150-153, 1959
5. Holborow, E. J., et al. Distribution of blood group A antigen in human tissues. Brit. J. Exper. Path. 41:430-437, 1960
6. Szulman, A. E. Histological distribution of blood group substances A and B in man. J. Exper. Med. 111:785-800, 1960
7. Idem. Histological distribution of blood group substances in man as disclosed by immunofluorescence. 11. H antigen and its relation to A and B antigens. J. Exper. Med. 115:977-996, 1962
8. Hayashi S, Noguchi K, Yagihashi A, Starzl TE, Iwaki Y. Expression of blood group antigen (A, B, H, Le(a), Le(b)) on liver allografts. Transplant Proc 1992: 24: 2567-2568
9. Lawler, S. D. Blood group substances in human milk. In European Society of Hematology. Proceedings of 7th-8th Congresses. 19591961. Edited by E. Neumark. 2 vol. Vol. 2. White Plains, New York: Phichig. A. J., 1960. Pp. 1219-1222
10. Denborough, M. A., Downing, It. J., and McCrea, M. G. ABO system in milk and saliva. Australasian Ann. Med. 16:320-325, 1967
11. Hostrup, H. A and B blood group substances in serum of normal subjects. Vox Sang. 7:704-721, 1962
12. Idem. A and B blood group substances in serum of newborn infant and fetus. Vox Sang. 8:557-566, 1963
13. King, J. S., Jr., Fielden, M. L., Goodman, H. 0., and Boyce, W. H. Total nondialyzable solids in human urine. X. Isolation and characterization of nonultrafiltrable material with blood group substance activity. Arch. Bi"hem. 95:310-315, 1961
14. Lundblad, A., and Berggard, 1. Isolation and characterization of urinary A and B agglutinogen fractions. Biochem. et biophys. acta 148:146-150, 1967
15. Lundblad, A. Two urinary oligosaccharides characteristic of A, and B secretors: isolation and partial characterization. Biochem. et biophys. acta 148:151-157, 1967
16. Lundblad, A. Two urinary oligosaccharides characteristic of A, and B secretors: isolation and partial characterization. Biochem. et biophys. acta 148:151-157, 1967
17. Szulman, A. E. Histological distribution of blood group substances in man as disclosed by immunofluorescence. Ill. A, B, and H antigens in embryos and fetuses from 18 mm in length. J. Exper. Med. 119:503-516, 1964
18. Idem. ABH antigens in human tissues and secretions during embryonal development. J. Histochem. 13:752-754, 1965. Franks, D. Antigens as markers on cultured mammalian cells. Biol. Rev. 43:17-50, 1968
19. SP Yip, Blood, 95:1487, 2000.
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