Structural Effects of Naturally Occurring Human Blood Group B Galactosyltransferase Mutations Adjacent to the DXD Motif

Human blood group A and B antigens are produced by two closely related glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) utilizes UDP-GalNAc to extend H antigen acceptors (Fucα(1–2)Galβ-OR) producing A antigens, whereas a galactosyltransferase (GTB) utilizes UDP-Gal as a donor to extend H structures producing B antigens. GTA and GTB have a characteristic 211DVD213 motif that coordinates to a Mn2+ ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the 211DVD213 motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the Bel variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to AweakB phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in kcat compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn2+ was disrupted by Arg-214 causing displacement of the metal by a water molecule. Kinetic characterizations of the M214T and M214V mutants revealed they both had GTA and GTB activity consistent with the serology. The crystal structure of the M214T mutant showed no change in DVD coordination to Mn2+. Instead a critical residue, Met-266, which is responsible for determining donor specificity, had adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity. Human blood group A and B antigens are produced by two closely related glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) utilizes UDP-GalNAc to extend H antigen acceptors (Fucα(1–2)Galβ-OR) producing A antigens, whereas a galactosyltransferase (GTB) utilizes UDP-Gal as a donor to extend H structures producing B antigens. GTA and GTB have a characteristic 211DVD213 motif that coordinates to a Mn2+ ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the 211DVD213 motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the Bel variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to AweakB phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in kcat compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn2+ was disrupted by Arg-214 causing displacement of the metal by a water molecule. Kinetic characterizations of the M214T and M214V mutants revealed they both had GTA and GTB activity consistent with the serology. The crystal structure of the M214T mutant showed no change in DVD coordination to Mn2+. Instead a critical residue, Met-266, which is responsible for determining donor specificity, had adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity. The ABH histo-blood group antigens are inherited carbohydrate structures on erythrocytes and other cells. Individuals with blood group A carry at least one allele encoding a 3-α-N-acetylgalactosaminyltransferase (GTA), 4The abbreviations used are: GTA, 3-α-N-acetylgalactosaminyltransferase; GTB, 3-α-galactosyltransferase; MOPS, 4-morpholinepropanesulfonic acid. whereas those with blood group B have at least one allele giving rise to an 3-α-galactosyltransferase (GTB). GTA utilizes UDP-GalNAc as its donor substrate and the Fucα(1–2)Galβ-OR structure as an acceptor, where R is a glycoprotein or glycolipid (Fig. 1). Thus GTA synthesizes the blood group A antigen, GalNAcα(1–3)[Fucα(1–2)]Galβ-OR. GTB uses UDP-Gal as a donor for the same H acceptor, yielding the B antigen, Galα(1–3)[Fucα (1–2)]Galβ-OR (Fig. 1). GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268 (1Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (908) Google Scholar, 2Yamamoto F. McNeill P.D. J. Biol. Chem. 1996; 271: 10515-10520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Therefore, they constitute a useful model system for glycosyltransferase structure-function studies. Kinetic studies demonstrated that residue Leu/Met-266 is critical for differentiating between the UDP-GalNAc donor for GTA and the UDP-Gal donor of GTB (3Seto N.O.L. Compston C.A. Evans S.V. Bundle D.R. Narang S.A. Palcic M.M. Eur. J. Biochem. 1999; 259: 770-775Crossref PubMed Scopus (88) Google Scholar). This observation was understood upon the determination of their crystal structures (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google Scholar), where the larger side chain of Met-266 in GTB blocks the N-acetyl group of the donor sugar from binding in the active site. In GTA the smaller side chain of Leu-266 allows the N-acetyl group of the UDP-GalNAc donor into the active site. Glycosyltransferases have been categorized into 87 different families based on amino acid sequence similarity (5Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar, 6Davies G.J. Gloster T.M. Henrissat B. Curr. Opin. Struct. Biol. 2005; 15: 637-645Crossref PubMed Scopus (241) Google Scholar, 7Breton C. Snajdrova L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: 29R-37RCrossref PubMed Scopus (504) Google Scholar). The blood group-synthesizing GTA and GTB are classified as retaining, metal-dependent, family 6 enzymes. However, despite the enormous sequence diversity of glycosyltransferase enzymes, only two major structural folds have been observed, the GT-A fold and the GT-B fold (6Davies G.J. Gloster T.M. Henrissat B. Curr. Opin. Struct. Biol. 2005; 15: 637-645Crossref PubMed Scopus (241) Google Scholar, 7Breton C. Snajdrova L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: 29R-37RCrossref PubMed Scopus (504) Google Scholar, 8Hu Y. Walker S. Chem. Biol. 2002; 9: 1287-1296Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The blood group GTA and GTB enzymes both exhibit the GT-A structural fold (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google Scholar), which is characterized by one N-terminal domain containing a Rossmann fold responsible for the recognition of donor and one C-terminal domain separated by a cleft containing the active site. Enzymes with a GT-A fold frequently require metals (Mn2+,Mg2+) for activity (7Breton C. Snajdrova L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: 29R-37RCrossref PubMed Scopus (504) Google Scholar) and have a characteristic DXDor equivalent motif that coordinates to the phosphate in the donor via the metal (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google Scholar, 7Breton C. Snajdrova L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: 29R-37RCrossref PubMed Scopus (504) Google Scholar, 9Gastinel L.N. Bignon C. Misra A.K. Hindsgaul O. Shaper J.H. Joziasse D.H. EMBO J. 2001; 20: 638-649Crossref PubMed Scopus (179) Google Scholar). In GTA and GTB the motif is defined by residues 211DVD213. The Asp residues coordinate a Mn2+ ion, which in turn coordinates to the phosphates of the UDP-sugar donor. Both aspartate residues bind to the Mn2+ including a bidentate coordinate bond by Asp-213 (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google Scholar). Mutagenesis studies have shown that the DXD motifs in GT-A fold glycosyltransferases are not functionally equivalent. In some cases the DXD motif is important for enzyme activity but not for binding of the sugar nucleotide (10Munro S. Freeman M. Curr. Biol. 2000; 10: 813-820Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 11Gøtting C. Muller S. Schøttler M. Schøn S. Prante C. Brinkmann T. Kuhn J. Kleesiek K. J. Biol. Chem. 2004; 279: 42566-42573Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 12Li J. Rancour D.M. Allende M.L. Worth C.A. Darling D.S. Gilbert J.B. Menon A.K. Young Jr., W.W. Glycobiology. 2001; 11: 217-229Crossref PubMed Scopus (28) Google Scholar), whereas in other instances it is important for both nucleotide binding and activity (13Busch C. Hofmann F. Selzer J. Munro S. Jeckel D. Aktories K. J. Biol. Chem. 1998; 273: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 14Gulberti S. Fournel-Gigleux S. Mulliert G. Aubry A. Netter P. Magdalou J. Ouzzine M. J. Biol. Chem. 2003; 278: 32219-32226Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The only exception reported to date is leukocyte type 2 6-β-N-acetylglucosaminyltransferase, which displays the GT-A fold type but lacks a DXD metal ion binding site. Instead, the role of the metal ion is served by two basic amino acids, Arg-378 and Lys-401 (15Pak J.E. Arnoux P. Zhou S. Sivarajah P. Satkunarajah M. Xing X. Rini J.M. J. Biol. Chem. 2006; 281: 26693-26701Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The subgroups of the ABO blood group system were originally characterized based on their serological behavior in red blood cells and plasma typing. Subsequently, individuals with anomalous blood grouping results were further categorized following DNA sequencing of their ABO genes (16Olsson M.L. Irshaid N.M. Hosseini-Maaf B. Hellberg Å. Moulds M.K. Sareneva H. Chester M.A. Blood. 2001; 98: 1585-1593Crossref PubMed Scopus (166) Google Scholar, 17Seltsam A. Hallensleben M. Kollmann A. Blasczyk R. Blood. 2003; 102: 3035-3042Crossref PubMed Scopus (124) Google Scholar, 18Roubinet F. Janvier D. Blancher A. Transfusion. 2002; 42: 239-246Crossref PubMed Scopus (35) Google Scholar). These subgroups include variants of blood group B, in which individuals with mutations adjacent to the 211DVD213 motif, M214V or M214T, have been classified as AweakB subgroups (19Deng Z.H. Yu Q. Wu G.G. Lian Y.L. Su Y.Q. Li D.C. Wang D.M. Zhang S.Y. Vox Sang. 2005; 89: 251-256Crossref PubMed Scopus (19) Google Scholar). The erythrocytes in these cases react weakly with some anti-A reagents as well as with all anti-B sera, suggesting that the blood group glycosyltransferase exhibits GalNAc as well as Gal transferase activity. A third mutation, M214R, has been classified as aBel subgroup (20Ogasawara K. Yabe R. Uchikawa M. Saitou N. Bannai M. Nakata K. Takenaka M. Fujisawa K. Ishikawa Y. Juji T. Tokunaga T. Blood. 1996; 88: 2732-2737Crossref PubMed Google Scholar). The amount of B determinant on these erythrocytes is very low, so that they do not agglutinate when incubated with monoclonal or polyclonal anti-B reagents. However, upon incubation with polyclonal blood grouping reagents, these cells will adsorb and subsequently elute measurable quantities of anti-B. Also, these individuals typically lack anti-B in their sera. Here we report the expression, kinetic analysis, structure determination by single crystal x-ray diffraction, and structurefunction correlations of variant GTB enzymes corresponding to the naturally occurring mutants, M214T, M214R, and M214V, which all have mutations adjacent to the DXD motif. Each of these constructs is based on the same synthetic gene that has been previously used for high level expression of soluble GTB in Escherichia coli (3Seto N.O.L. Compston C.A. Evans S.V. Bundle D.R. Narang S.A. Palcic M.M. Eur. J. Biochem. 1999; 259: 770-775Crossref PubMed Scopus (88) Google Scholar, 21Marcus S.L. Polakowski R. Seto N.O.L. Leinala E. Borisova S. Blancher A. Roubinet F. Evans S.V. Palcic M.M. J. Biol. Chem. 2003; 278: 12403-12405Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Materials—Oligodeoxynucleotides, UDP, UDP-Gal, and UDP-GalNAc were obtained from Sigma. The acceptor substrate (Fucα(1–2)Galβ(CH2)7CH3) was a kind gift from Prof. Ole Hindsgaul. Sep-Pak C18 reverse-phase cartridges (Waters), UDP-[6-3H]GalNAc (50μCi/mmol) and UDP-[6-3H]Gal(250μCi/mmol) (American Radiolabeled Chemicals), Ecolite(+) liquid scintillation mixture (ICN), Vivaspin protein concentrators (Vivascience AG, Hannover, Germany), and restriction enzymes (Promega) were obtained from the suppliers indicated. All other chemicals used were of at least reagent grade. Site-directed Mutagenesis—A QuikChange II XL site-directed mutagenesis kit (Stratagene) was used to generate mutants. The PCR step was performed in a thermocycler Peltier Thermal PTC-200 (Bio-Rad). The plasmid pCWΔlac vector harboring the –10GTB (amino gene was used as a The were to the The used for mutagenesis were as and The that were are The PCR mixture was with of for at to plasmid The were into E. coli and sequencing was performed by mutants were into E. coli cells for in E. enzyme variants were in E. coli cells and with a an step by a as previously (3Seto N.O.L. Compston C.A. Evans S.V. Bundle D.R. Narang S.A. Palcic M.M. Eur. J. Biochem. 1999; 259: 770-775Crossref PubMed Scopus (88) Google Scholar, 21Marcus S.L. Polakowski R. Seto N.O.L. Leinala E. Borisova S. Blancher A. Roubinet F. Evans S.V. Palcic M.M. J. Biol. Chem. 2003; 278: 12403-12405Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). were with a M.M. M. Hindsgaul O. J. Scopus Google Scholar). In a Sep-Pak reverse-phase was used to that were produced when was from a donor to a acceptor, M.M. M. Hindsgaul O. J. Scopus Google Scholar). were performed at in a of containing substrate and enzyme in and different of donor and acceptor were and the were with no of the substrate in the the UDP-Gal donor, the for all mutants were at acceptor, and the for the acceptor was at UDP-GalNAc, the was at acceptor for the M214T mutant and at acceptor for the M214R and M214V mutants. for the acceptor was at UDP-GalNAc for M214V and M214R and at acceptor for The kinetic kcat and were obtained by analysis of the with the The for the wild type GTB, M214T, and M214V were as in the kinetic the was and the substrate were and for donor and for acceptor or 2 for both A of enzyme is the amount that of substrate to in were the with as a protein GTB mutant structures were to the GTA and GTB enzymes (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google Scholar). were on a at of and between and for and with Biol. 1999; PubMed Scopus Google Scholar). were produced by a to x-ray with levels of The were and at a of a crystal All structures were by with wild type GTB as a model A. A. J. Scopus Google Scholar) and were Biol. PubMed Scopus Google Scholar) in the Biol. PubMed Scopus Google Scholar). of were to B of variants of GTB in blood banking with mutations at in the of the 211DVD213 motif were These enzymes were all as soluble of the domain (amino and in E. The of the constructs by and on a was in all The of protein were for the M214T for the M214V and for the M214R and were for enzyme at a high of the alternate substrate 1). the M214R mutants, the kcat for both UDP-Gal and UDP-GalNAc was as compared with the wild type GTB, which exhibits a kcat of for UDP-Gal and for UDP-GalNAc 1). The binding of UDP-Gal and acceptor was for mutant with an in for UDP-Gal from for the wild type enzyme to for the The acceptor had been from to Individuals with mutation generate low quantities of B antigen on their red blood consistent with the results obtained in blood banking where the mutation was in the B allele of an was and categorized as Bel (20Ogasawara K. Yabe R. Uchikawa M. Saitou N. Bannai M. Nakata K. Takenaka M. Fujisawa K. Ishikawa Y. Juji T. Tokunaga T. Blood. 1996; 88: 2732-2737Crossref PubMed Google Scholar). This allele was originally by the and to the 2004; PubMed Scopus Google and for wild type and mutant in a The M214T mutant has both glycosyltransferase A and glycosyltransferase B activity 1). Kinetic analysis of the mutant the UDP-Gal revealed that the kcat for UDP-Gal was whereas the kcat for the UDP-GalNAc compared with wild type GTB 1). The mutation a on the binding of the acceptor substrate in an in for acceptor 1). This in the expression of levels of B antigen on and low levels of A antigen, which is in with the A mutation in M214T was in the B allele to the 2004; PubMed Scopus Google of an as AweakB (19Deng Z.H. Yu Q. Wu G.G. Lian Y.L. Su Y.Q. Li D.C. Wang D.M. Zhang S.Y. Vox Sang. 2005; 89: 251-256Crossref PubMed Scopus (19) Google Scholar), despite the of an A allele in with for the A and the B the M214V mutant was no on the kcat for the UDP-GalNAc, whereas the kcat for UDP-Gal was The were in a as the M214T enzyme 1). The activity is not of of the M214V The mutant of its activity incubation at for which is with that of wild type GTB. The kinetic expression of B but the low kcat for UDP-GalNAc is not consistent with the AweakB in individuals B were in M214V in GTB (19Deng Z.H. Yu Q. Wu G.G. Lian Y.L. Su Y.Q. Li D.C. Wang D.M. Zhang S.Y. Vox Sang. 2005; 89: 251-256Crossref PubMed Scopus (19) Google Scholar). Hosseini-Maaf and M. L. This allele was to the 2004; PubMed Scopus Google Scholar). were at both low or and high of low donor the activity of M214V was wild type GTB, whereas at high donor wild type GTB was M214V substrate are not the AweakB is consistent with enzyme low substrate activity for wild type and mutant blood type B 2 2 the activity was low to the activity was low to in a M214R and M214T mutants were but not be obtained for the M214V The M214T mutant was in with UDP, but was not for were at of and of the and results for the GTB mutants and are shown in were to a from to with from to and an of and All structures showed the of the chain with the exception of the (amino and the C-terminal amino acid both of which were in the structures (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google and results for of GTB mutants and in the highest in the highest of were for bond in the highest of were for in a The 211DVD213 motif in the blood group A and B glycosyltransferases coordinates a Mn2+ ion, which in turn binding (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google Scholar). is not that the mutations in important of the enzyme but it is that they enzyme substrate specificity. The very low substrate for the M214R mutant be understood upon of its crystal In the wild type the two aspartate residues coordinate a Mn2+ in the mutant enzyme Arg-214 with and Asp-213 to a water in the Mn2+ binding which not donor binding (Fig. The of a water a low occupancy Mn2+ ion is not only by the bond to the group of the residue but by the between and Asp-213 and the water and between these residues and the Mn2+ ion in the wild type enzyme and a in the anomalous be for a Mn2+ no was adjacent to the DVD motif. the of the bond from Arg-214 to the water is at Å. between are by S. S. J. 2005; PubMed Scopus Google Scholar), and is a the water as it between Arg-214 and the two aspartate residues of the 211DVD213 motif. Arg-214 not of the active site critical residues to be in donor recognition and was no of the mutation enzyme by the active as in the blood group mutant enzyme Borisova S.N. Seto N.O.L. Blancher A. Evans S.V. Palcic M.M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). to a structure of M214R in with were of Mn2+ and used for the wild type but for or was not M214T showed both GTA and GTB activity 1). The mutation of the to the smaller in GTB in the of a adjacent to the active site. The side chain of by into in a in the active site the critical residue Met-266, with the that the side chain of residue be to at least different (Fig. all of which to the of side chain in J. 2003; PubMed Scopus Google Scholar). The major conformation with (Fig. one of the is to that for Met-266 in the wild type enzyme and the other into the donor binding site The anomalous a at corresponding to the in the high occupancy The of these were to and which for The alternate conformation of Met-266 with highest occupancy the side chain with and a in the active site (Fig. the coordinates of from the M214T structure in with UDP, the of UDP-Gal and UDP-GalNAc be into the binding site (Fig. The conformation for UDP-Gal is in with the structure of donor by J. B. A. T. B. H. Palcic M.M. T. J. Chem. 2006; PubMed Scopus Google Scholar). This that the conformation of Met-266 the active site to accommodate the N-acetyl group of GalNAc in the donor binding site (Fig. in blood group A wild type GTB the of and Met-266 to M214T the of and Met-266 to The conformation of Met-266 opens up the active site. M214T with The larger UDP-GalNAc, be by the high occupancy conformation of Met-266 and not the low wild type conformation are by The Gal and GalNAc of the are shown with of the M214V mutant not However, it is to some structure-function the change in side chain from the wild type to is in and to the M214T mutation The smaller side chain the of from Met-266 and a active site recognition that and subsequently the larger A donor. of donor substrate upon mutagenesis have been reported for other glycosyltransferases B. E. Biochem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Curr. Opin. Chem. Biol. 2006; 10: PubMed Scopus Google Scholar). These include mutants of with activity with a single amino acid B. E. 2005; PubMed Scopus Google Scholar), and UDP-GalNAc transferase activity with a mutation B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The donor of B has been to by a mutation based on with a related transferase and T. T. Aktories K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). A was to an enzyme with and transferase activity by a mutation M. S. N. Netter P. Magdalou J. Fournel-Gigleux S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). A galactosyltransferase was to a with a mutation A. Y. S. T. Biochem. 2004; PubMed Scopus Google Scholar). is of donor to a mutations is glycosyltransferases and GTA and GTB are other glycosyltransferases to In mutations of residue in the blood group B enzyme were to as they are adjacent to the 211DVD213 motif (4Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (202) Google Scholar, B. E. Biochem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), which is for donor However, they have been shown to be to the and of the active site donor recognition M214R very substrate as the residue on the of the 211DVD213 motif to bind Mn2+ no to donor sugar M214T and M214V enzymes exhibit both glycosyltransferase A and B which be by the the M214T and the M214V mutation to a of the active site residues to the binding and of the A donor. In the of M214T and M214R, the serological results are in high with the a its the ABO blood group system is one of the and model for studies of correlations as well as the structure-function of

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