Biochemistry and biosynthesis of ABO blood group system
ABO blood group antigens are surface markers on the red cell, and consist of proteins and carbohydrates attached to lipids or proteins. ABO antigens are widely expressed on the membranes of red cell and tissue cell as well as, in the saliva and body fluid. ABH antigens are also detected on endothelial cells and epithelia from the lung and the gastrointestinal tract and also on the lining of the urinary and reproductive tracts. The formation of ABO antigens are not directly encoded by genes. However, ABO antigens are synthesized by enzymatic reactions catalyzed by specific glycosyltransferases A and B enzymes that respectively add N-acetyl galactosamine and galactose to a precursor chain, the H antigen, by an identical α1-3glycosidic. Initial steps for biosynthesis of ABO antigens is began by addition of L-fucose on terminal galactose (Gal) of type 1 precursor, resulting in H antigen. Subsequently, the A antigen is synthesized by enzymatic reactions catalyzed by glucosyltransferase (α1,3-N-acetyl-D-galactosaminyltransferase) which adds N-acetyl galactosamine to a precursor chain, the H antigen, and the B antigen is synthesized by enzymatic reactions catalyzed by α1,3-D-galactosyltransferase that adds galactose to H antigen (Hakomori, 1981).
Both glycosyltransferase enzymes A and B (GTA and GTB) are membrane-bound proteins of about 330 amino acids that differed from each other by only four amino acids. Both enzymes would recognize the same precursor, H antigen acceptor but they recognize two different monosaccharide donors: uridinediphosphate-GalNAc for GTA, and uridinediphosphate-galactose for GTB. Two of the four amino acids that differ between GTA and GTB are located in the active site of both GTA and GTB enzymes. The larger residues methionine 266 and alanine 268 and the smaller residues leucine 266 and glycine 268 are located in active site of GTA and GTB enzymes respectively. These amino acids that differ between GTA and GTB were proposed to must serve to recognize the two different monosaccharide donors: uridinediphosphate-GalNAc for GTA and uridinediphosphate-galactose for GTB.
Genetics of the ABO blood group system
The genetics underlying the structures of ABO antigens are more complex because ABO antigens are generated by sequential action of a number of enzymes. In addition, the same phenotype is sometimes produced from different genotype and also different phenotypes are sometimes produced from the same genetic variant. Hence, prediction of ABO phenotype from genotype is difficult. Two mechanisms are considered to be responsible for genetic diversity and diversification at the ABO locus: point mutations and genetic recombination.
The glycosyletranseferase gene
The ABO genes do not directly code for the production of ABO antigens, but rather produce specific glycosyltransferases that add either N-acetyl galactosamine or galactose to the H antigen then converting it into the A or B antigen respectively. Remarkably, the difference between the A and B glycosyltransferase enzymes is only four amino acids. The gene encods ABO antigens is located at the long arm of chromosome 9q34. Moreover, ABO gene consists of at least 7 exons, and the coding sequence in the seven coding exons spans over 18-20 kb of genomic DNA. There are six introns with sizes ranging from 554 to 12982 bp. The last exons 6 and exon 7 of ABO gene consist 823 of 1062 bp of the transcribed mRNA, covers 77% of the whole encoded protein or 91% of the catalytic domain of the ABO glycosyltransferase. The amino-terminal cytosolic domain, a trans membrane domain, the stem region, and the remainin 9 % of catalytic domain of the ABO glycosyltransferase are encoded by exon 1, exon 2, exon 3, exon 4 and exon 5. The exon 6 consists a large number of single nucleotide deletion of O alleles and responsible for the loss of the activity of the enzyme. Beside, exon 6 consists of the first of the seven nucleotide substitutions which distinguish the A and B transferases. The exon 7 consists of the other six nucleotide substitutions which distinguish the A and B transferases. Specificity of glycosyltransferase A or B enzymes are determined by single nucleotide substitution that results in amino acid substitution at two sites (residue 266 and 268). There are four common alleles of ABO gene; A1, A2, B and O. There are also numerous others alleles of ABO gene which are resulting in a number of amino acids changes abolish or weaken the enzymatic activity of ABO glycosyltransferases. The studies on the ABO gene polymorphisms were not only focused on the structural gene but also on the number and positioning of introns, the promoter and enhancer regions of the genes. In 1997 the characterization of enhancer-active minisatellite sequences (approximately 4 kbp upstream from the start codon)that required binding of the transcription factor CBF/NF-Y for ABO mRNA transcription to occur in a gastric cancer cell line was reported.
The A alleles
A number of ABO blood groups variants have been reported, with approximately 250 different alleles registered in the Blood Group antigen gene Mutation database (BGMUT). Blood type A with a normal quantity of antigen is called A1, and is distinguished from subgroups. Subgroups are classified by the quantity of A antigen, and the amount of A antigen decreases in the order A1, A2, A3, Ax, Aend, Am, Ael. There are about 20 more subgroups of A blood type have been identified. A1 subgroup (A101) is the most common A phenotype and equals approximately 80% of the entire A blood type of population. The cDNA (the A1allele, (A101) encoding the N-acetylgalactosaminetransferase was cloned andsequenced by Yamamoto,et al. (1990) and is taken as the reference allele or consensus sequence on which all other variant of ABO genes are compared. In 1990, Yamamoto et al. identified most common A1 variants, 467C>T (A102) in Asian population. Next to A1 allele, the A2 allele (A201) is the most common allele of A blood type. A2 allele has a substitution mutation in exon 7, 467C>T (Pro156Leu) and deletion mutation of one of the three cytosines at nucleotides 1059 to 1061 which results in an extension of the reading frame by 64 nucleotides and production of transferase with an extra 21amino acids at its C-terminus, causing a less effective enzyme.
In 1995, Olsson & Chester identified seven nucleotide substitutions (297A-G, 526C-G, 657C-T, 703G-A, 796C-A, 803G-C and 930G-A throughout exon 6 and 7) which separated B1 (B01) allele from A1 allele (A101). Four nucleotide substitutions 526C-G, 703G-A, 796C-A and 803G-C are translated into different amino acid substitutions Arg526Gly, Gly703Ser, Leu796Met and Gly803Ala respectively and they discriminate GTA from GTB. The amino acid changes at 266 (Leu>Met) and 268 (Gly>Ala) are the most critical for B enzyme activity.
The most common O1allele was shown to be identical with A1 allele except for nucleotide deletion in exon 6 at 261delG, which leads to a stop codon and a truncated, inactive enzyme . There are many other variants of O alleles not having this deletion but still causing inactive enzymes.