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From www.bloodjournal.org by guest on June 14, 2017. For personal use only. Identification of Genotypes of Blood By Akira The human blood carbohydrate groups chains galactosyltransferase cell surface. to both but determine or BB existence of CRM separating CRM but T certainty not governing active the AO by these HE HUMAN be determined group 0 88 or was but plasma. 88 should transferase responsible cell (B-enzyme) for producing surfaces, with in blood A and both AA or AO, since BB or BO) neither activities heterozygote. anti-A-enzyme B-enzymes and cally inactive that but protein component zygous 40 contains A- and A,- that CRM and properties genotypes, certainty by such of the plasma. indeed, can hetero- Blood Homozygous parents. A,A, with and Heterozygous pedigree. of CRM with Assay A persons B substances their group and or 80 BB nonallelic was inactive. profiles plasma. plasma, genotypes and for the demonstrated blood ABO by groups heterozygous model of In addition. was of the red family A-enzyme Immunoneutralization AO and on the examining blood common of an enzymatically in 0 plasma any and B-Enzyme ofA- The transferase ration can AO locus. be and The 80 genes of the nucleotide from natural A-enzyme M NaCI, mM NaN3, acceptor.6 The contained 2 mM The (‘H, 0.2% assay, bovine and reaction ml). The 25 was reaction mixture was also reports with sugars, and less than subtracted blood Tris-CI, group the pH in the that and enzyme 7.0, was N-acetylgalactosamine was UDP-galactose was New England 98% Nuclear and milk as with Corp. previously Biogel as determined Purity by its sugar at with 0.01 reported.5 Chemical Co. UDP- purchased was from composition. was prepared further from purified preparation M UDP- previously was as -60#{176}C. (galactose-6-3H) and of the purified albumin. Fucosyllactose described,9 P-2. enzymes) form I -3H) UDP-galactose from Amersham purchased human (galactosamine- enzyme partially diluted as control as mixture. serum Sigma the purified reaction were from hydrolysis plasma of partially bovine 2 fucosyllactose of crude synthesized purchased N-acetylgalactosamine catalytic in concentrated 1% strip (total by a scintillation minus appropriately containing as aliquot withwater radioactivity complete stored used 37#{176}C.An eluted B-enzymes and used to an arrow-shaped of case of the A- reported,t8 use, Before 3% from as a buffer. and case I the was mixture The For 0.5 MCi) for spontaneous in the X-lOO, was 16 hr at and reaction Triton enzyme. 6.5, measured a control. 10% and was the as than (less eluate UDPMnCI2, (3H, applied (2 x 7 cm) to correct incubated mixture for was of the nucleotide pH incubated paper 6.5. MI. for 15 mM 0.25% pH galactose = 100 25 MM ATP, albumin, imidazole, mixture In order of the UDP mixture, I mM incorpoan analog fucosyllactose, buffer, mM radioactivity counter. serum the fucosyllactose, reaction MCi), cacodylate 25 MM donor, 0.5 by measuring into sugar 40 mM B-enzyme a sugar sugar assay N-acetylgalactosamine 0.15 Activity were assayed activities of sugar The METHODS not change antiserum plasma group during against previously.5 The reconstituted Vol. 55, A- and (ACD the BO of A-enzyme was 1 (January), salted pH 1980 at was out 7.0, with and from ofAB were by was activities period obtained offspring anticoagulant) storage purified were are blood samples were identified B-transferase in 0.01 M Tris-CI, No. samples B who genotypes and antibody blood A, and A,O whose Fresh blood BB phenotypes individuals The Blood, plasma. AA to group was by gel better Sepharose 4B than (lot no. Samples individuals from existence filtration AND 0 The existence Thus, previously approaches. MATERIALS B persons. in homozygous was in addition This article be identified A and of heterozygous source, to the active A- or B-enzyme, while homozygous AA and BB plasma should not contain CRM. Therefore, it should be possible to determine genotypes of blood group A and B persons by examining the immunobiochemical that the without of DEAE-cellulose an enzymaticross-reactive It is expected plasma impossible. (20 MI) of the the homozygote it was found that 0 plasma contained immunologically BO are red B persons, both been in those the group group neutralized (CRM).5 and blood distinguish Recently, antibody in blood A and nor can red (A- without examingenotypes (i.e., of phenotype agglutinability B-enzyme from the absent impossible, to determine A and The attached in blood allelic. group B persons B substances on enzymes 0 persons.’ It has been ing their family pedigree, not known to residues and B residues producing prepared. is incompatible be truly for It has was The galactosyl- persons responsible chromatography. methods. enzyme) A glycosyl (A-enzyme) demonstrated affinity of the group terminal of group from plasma, B) by blood groups (ABO) are by the terminal glycosyl blood 80) (CRM). attached to common carbohydrate chains cell surface. N-Acetylgalactosaminyltransferase in the persons. A-enzyme 80 and and AA are against biochemical expression by B persons distinguished and (A determined or AO, clearly enzyme group AA antibody were in homozygous blood group protein plasma be in blood (i.e.. rabbit in heterozygous and with plasma absent and to A and Yoshida N-Acetylgalactosaminyltransferase in blood cross-reactive AA identified known surface. genotypes immunologically homozygous cell enzymes to homogeneity. purified are red (B-enzyme) with pedigree. (ABO) of the Group obtained their stored 2 mo. as at Submitted Address - Biochemical E. Duarte described 2 M (NH4)2S04 and I 5#{176}C. Department of Center, in part by Biochemical Duarte. Genetics, City of Hope Calif USPHS Grant HL-20301 and NSF Grant FJ-5078. did Rabbit the Medical 60#{176}C. plasma prepared stored - From National Supported family at in the least x AB © 1980 July 2. 1979; reprint requests to City ofHope Genetics, Road, Duarte, by Grune accepted Calif & Stratton, September Akira 21, Yoshida. National Medical 1979. Department Center of /500 91010. Inc. 0006-497I/80/550l0021$01.00/0 119 From www.bloodjournal.org by guest on June 14, 2017. For personal use only. 120 AKIRA Table 1 . Blo od Group A- and B-Enzyme Activity Enzyme in Plasma YOSHIDA 100 Activity 90 Sample Genotype A B AA-1 AA-2 AA-3 A,A 17.2 A,A 9.2 A,A 17.1 AO-1 A,O 8.6 A,O AO-2 Phenotype - 7.2 A, mean (9 samples) range 6.44, t; 80 Q) 70 0 - E A 60 , c: w 2.8-16.8 A Q) BB-1 88 - BB-2 BB - BO-1 80 - BO-2 BO - Phenotype 15.3 range composition 50 of the reaction The enzyme actosamine activity under 10) was 4B (1 mol pl. used from Sigma was prepared gel) assay. of sugar described Co. Plasma and B-Enzyme A- A- and AND B-enzyme described.’0 plasma activities plasma nearly were in the twice that homozygous and in Table I. I and AA-3 of heterozygous plasma, that of the heterozygous The A- and B-enzyme and B plasma From the known of the differed genotype samples were higher than that of the B-enzyme activity (BB-2) was similar to of phenotypes widely among frequencies, the most, A, samples. if not all, to be heterozygous A,O The plasma Tris-CI, pH plasma (or albumin) 40 anti-A-enzyme ofPlasma A- and Antibody samples 7.0, at plasma sl, bated at 4#{176}C for an assay of the immunoneutralization A- and B-enzyme were more strongly were B-Enzyme dialyzed diluted with was mixed with antibody (totaling An 1% various 50 0.01 of M aliquot bovine of serum amounts of sl) and incu- 5 hr with shaking, then subjected remaining transferase activity. profiles indicated that activities neutralized 50 d) to an assay with homozygous by the same 4 5 1% bovine and of remaining antibody. the 6 7 8 9 10 ui serum AA-1 at and albumin 4’C to The the plasma amount of for A-enzyme to AA-3 plasma result 5 hr. activity. in AA The of then Prior were to diluted approximately the same activity as that of AO-1 plasma. The values represent mean values of duplicate analysis taking the control (without antibody) as 100. (C) AA-1; (A) AA-2; (U) AA-3; (C) AO-1; (M AO-2; (t) mixture (1 :1 ) of AA-1 and 0 plasma. antibody than those and 0 plasma heterozygous of heterozygous Existence were plasma. plasma has Inactive known adsorbed A-enzyme uridine that with Sepharose 4B A,O plasma, and 0 Sepharose 4B column plasma, column with was washed (CRM) but not A,-enzyme Sepharose can be 5’-disphosphate.t Utilizing these adsorption heterozygous AO and BO A,- and B-enzymes. Aliquots of homozygous I but Protein AO and BO Plasma AA and BB Plasma been completely (Figs. profiles of a plasma and 0 BB- I plasma to that of corresponding I and 2). Cross-Reactive in Heterozygous in Homozygous It similar (Figs. of Enzymatically be adsorbed with with UDP-Sepharose against 4#{176}C overnight. 4 incubated (totaling adsorbed containing or BO. Neutralization by Anti-A-Enzyme I antibody Immunologically plasma. activities expected 3 I and 2). The immunoneutralization mixture (1:1) of homozygous AA-l plasma and a mixture of homozygous but the activity of homozygous AA-2 was only slightly higher than that of heterozygous plasma. Similarly, the B-enzyme activity of one of the homozygous plasma (BB-l) was distinctively two heterozygous samples, but of another homozygous plasma 2 I of A-enzyme in homozygous by anti-A-enzyme antibody. mixed with various amounts with samples are presented of homozygous AA- 1 I I Fig. 1 . Immunoneutralization and heterozygous A0 plasma dialyzed plasma. 40 zl. was mixing RESULTS Activities activities heterozygous The enzyme I Antibody, UDP-Sepharose as previously I into in the text. Chemical I The (N-acetylgal- transferred U . c: 1 7.5 subjected EXPERIMENTS A Q) 10.6. 5.0- enzyme for B-enzyme) conditions U 20 in the text. as percent and galactose obtained for was described expressed the assay UDP/ml was mixture was A-enzyme for fucosyllactose I 7C-Ol plasma, . 8.8 mean of 0 30 10.0 B aliquot N 9.4 (7 samples) An U 40 eluted 4B, with B-enzyme can be and the a buffer cannot 4B, but it can be adsorbed (unpublished observation). properties, CRM present in plasma was separated from (0.15 A,A, plasma, heterozygous 5 ml, were placed on a ml bed volume), and the I ml of 0.1 M cacodylate buffer, EDTA. pH 7.0, containing 2 mM MnC12 and I mM The eluate was dialyzed against 0.01 M Tris- Cl, 7.0, pH containing 5% glycerol and I mM EDTA, From www.bloodjournal.org by guest on June 14, 2017. For personal use only. GENOTYPE IDENTIFICATION OF ABO 121 SYSTEM 100 matography >% (final go :t 80 ci, 70 E so that 60 Lu The I ml) protein was used 4 N 30 . 0 inactivate A obtained 20 0) not A 10 :D I 1 2 3 1 1 1 4 5 6 Antibody , ii I.-. I I 7 8 neither assay of adsorbed respectively, (NEP) obtained A- nor B-enzyme react 10 I and B-enzymes. from homozygous A,A, with the and retained B-enzymes J___* 9 protein had A- solution I nonenzyme treatments solution for to by the activi- ty. When the antibody solution was incubated with NEP obtained from 0, heterozygous A,O, and BO plasma, the treated antibody solution lost its ability to t 0) :s the above 50 . above. of about CRM activity. The A- and B-enzymes were completely Sepharose 4B or to UDP-Sepharose 4B, .4- U as described volume antibody, its capacity (Table heterozygous 2). A,O BO Fig. 2. Immunoneutralization of B-enzyme in homozygous BB and heterozygous 80 plasma by anti-A enzyme antibody. The dialyzed plasma. 40 p1. was mixed with various amounts of antibody (totaling 50 gil) and incubated at 4’C for 5 hr. then subjected to an assay of remaining B-enzyme activity. Prior to mixing with antibody. the BB-1 plasma was diluted with 1 % bovine serum albumin to result in approximately the same activity as that of BO-1 plasma. The values represent mean values of duplicate analysis taking the control (without antibody) as 100. (C) BB-1 ; (A) BB-2; (C) 80-1 ; () BO-2; (C) mixture (1 :1) of BB-1 and 0 plasma. contrast, BB the for These and ma, contained CRM, plasma did not contain In and NEP plasma treated antibody inactivating results did A- and indicated plasma, as well but homozygous CRM. that as 0 A,A, plas- and BB DISCUSSION It has been known that the groups A and B subjects cannot agglutinability enzyme activities Table 2. of their differ Content of CRM blood. widely genotypes be identified Blood among in Homozygous and in Heterozygous blood by the group A and B individuals with and 88 A,A, and 80 A,0 of Plasma Plasma Percent of Enzyme Activity Neutralized by Antibody and placed 1.5 ml) containing EDTA. on a CM-Sephadex equilibrated with column 0.02 M 5% glycerol, 0.05 M The column was washed equilibration containing buffer and 0.08 M NaCI. then The (bed Tirs-CI, NaCI, with Exp. volume pH No. 7.0, and 2 ml I mM of the with 3 ml of the protein remaining buffer in the column was eluted from the column with 3 ml of the buffer containing 0.2 M NaC1 and precipitated with saturated ammonium sulfate. The protein (nonenzyme protein, NEP) pH 7.0, and adjusted then To was the to about performed I ml. on the separate homozygous were dialyzed 6.5, containing dialyzed volume CRM 2 3 final from NEP activity assay aliquots of BB and heterozygous BO plasma, 5 ml, against 0.025 M imidazole buffer, pH 15 mM MnCl2 and 0.15 M NaCI at 4#{176}C for 6 hr. The dialyzed plasma was UDP-Sepharose 4B (bed volume 3 ml) with the same buffer, and the mixture mixed with equilibrated was kept buffer Treated with NEP from AA-1 84 78 Treated with NEP from AO-1 <5 <5 with buffer with NEP from AA-2 80 Treated with NEP from AO-2 <5 Treated with buffer 92 Treated with NEP from <5 - <5 - Treated with NEP from 4 5 Treated with buffer Treated with NEP Treated with NEP from from 72 95 BB-1 66 95 BO-1 <5 <5 Treated with buffer - 88 Treated with NEP from BB-2 - 92 Treated with NEP from BO-2 - NEP (nonenzyme protein) Tris-CI, pH 7.0, as control). hr with shaking. An aliquot further analysis. chro- - 0 plasma stirring for 5 hr at 4#{176}C. A small glass filter was used to obtain clear supernatant, which was then treated with saturated ammonium sulfate. The protein precipitate was dialyzed against 0.01 M Tris-CI, ph 7.0, containing 5% glycerol and I mM EDTA, and subjected to column - phenotype of antibody. by CM-Sephadex 88 Treated Treated aliquot fractionation 92 with A, plasma was solution. B-enzyme, B-Enzyme 78 Treated pooled against 0.01 M Tris-CI, of protein solution was A CRM A-Enzyme Antibody 1 20 was zI, was prepared mixed with and the mixture of the mixture as described 11 in the text. An 40 MI of NEP (or 0.01 M was incubated (or Tris buffer at 4#{176}C for 5 as control), 20 0, was mixed with 1 0 MI of partially purified A B-enzyme and was incubated at 4#{176}C for 5 hr with shaking. The incubated enzyme was subjected to an assay described in the text. of A- and B-enzyme The results represent activity mean under values the conditions of duplicate From www.bloodjournal.org by guest on June 14, 2017. For personal use only. 122 AKIRA Allelic j A B 0 B !gu1atory Mde1 Non-Allelic !‘txlel Gene Mde1 A B 0 A B 0 A b a a b A ct A 6 A B B B B Fig. 3. Model for blood group These genes are assumed to 0 within several mosome the same phenotypes, and types, as reported fore, the genotypes their blood even group enzyme of the same nonallelic A, and geno- heterozygous and of homozygous different from BO plasma. A- and the existence of CRM plasma, but not was unequivocally ing CRM A-enzyme) and active enzyme and UDP-Sepharose After work this was by completed, However, tralization of their plasma, by examining the presence or absence plasma, as described in this article. Although the inheritance of blood answered. In the past, Furuhata with not both necessarily ABO chro- of Cis-AB AB x 0 is postulated originate crossing-over A and by to produce B genes a is theoretically assumes that structural are not allelic and that by allelic According to this produced in 0 subjects, shut and off not compatible with mendelian inheritance, and genes governing the ABO blood groups are generally considered to be allelic, the fundamental question, i.e., whether or not ABO genes are truly allelic, has not yet been of regulatory model, since silent. The only in 0, AA or BB plasma, be concluded that blood groups are truly genes for expres- both a, 13, and should A and not be B genes demonstrated heterozygous either It can their genes CRM are existence of with the regulabe rejected. should be produced AO and also in homozygous AA and/or present results indicate that CRM in their groups discovery offspring CRM in 0 plasma5 is incompatible tory gene model, so the model can In the nonallelic model, CRM directly, of CRM does is controlled 0. homo- more in possible even between true allelic genes. There are three conceivable models for ABO locus (ABO cistron), i.e., true allelic model, nonallelic model, and regulatory gene model (Fig. 3). The regu- sion conclusion. B subjects can immunoneu- and the AB In addition, latory gene model A- and B-enzymes samples of A,A2 plasma that all these plasma did confirming the above of phenotype A and certainty by examining profiles AO AA and BB by separat- additional pairs two sites, one for A, or not B.’2 Later, the true allelic by an Cis-AB chromosome Sepharose 4B (for 4B (for B-enzyme). one zygous BB plasma and four were examined. It was found not contain CRM, The genotypes be identified with in heterozygous in homozygous demonstrated involving B, or not i.e., base 9.’ challenged crossing-over. heterozygous AO and BO plasma contain CRM in addition to the active A- or B-enzyme, while homozygous AA and BB plasma do not contain CRM. In fact, was thousand ABO locus. be located parents.’3”4 Komai considered that the Cis-AB originated from crossing-over between A and B genes that were pseudoallelic but not allelic in the strict sense.’5 B-enzymes were more strongly neutralized in homozygous plasma than in heterozygous plasma. These results are most readily interpreted to mean that and BO plasma, model for expression, profiles distinctively AO one model activities. The immunoneutralization AA and BB plasma were those with by previous iflvestigators.” Therecannot be identified by measuring YOSHIDA BO subjects, but BB subjects. The does not exist in refuting the nonallelic the genes governing the model. ABO allelic. ACKNOWLEDGMENT The author Professor a ALA,, H. AA2, is indebted to Professor Taniguchi, and BB plasma and S. Miwa, Professor K. Dr. H. Yamaguchi, Bender for providing samples. REFERENCES I. mucosa 4. EurJ Race A 17:218, of ogy the and from B WM: blood gastric substance human specifications upon B character. J VM, Smith character. Yoshida ZG, Watkins associated Biochem A, J 109:315, Yamaguchi with WM: the An human a-N-acetylgalacblood 1968 YF, A H, H substance Partial purification 10:152, VM, a-galactosyltransferase and fluids. group Nagai Biochem M, V: Immunological homol- 9. Kobata Formation enzyme C, Watkins from WM: of blood back1979 group hog gastric mucosa. Biophys Res V, Kaplan a-N-acetylgalactosaminyl ovarian in human Dave J Biol A, genetic 54:344, by an a-N-acetylgalactosaminyltransof the glycosyltransferases. nyltransferase. Dave H: Blood 1969 Race and and determination. Schenkel-Brunner from Hearn glycosyltransferases (ABO) Biochem 8. group group Euri 7. Biochem Tuppy group blood blood ferase. An a-D-galactosyltrans- group of ground 1968 Hearn of human Nature 1970 D, Watkins with tosaminyl-transferase 5. conversion erythrocytes. H: Enzymes blood-group Biochem C, Ziderman associated 107:733, Enzymatic 6. H, Tuppy conferring ferase H: and of B into AB 1969 Schenkel-Brunner erythrocytes. 3. Tuppy 0 into A erythrocytes 223:1272, 2. H, Schenkel-Brunner human Commun BE, Yoshida I . Purification Chem Ginsburg 253:377, cyst epithelial 46:948, linings 1972 A: Human blood of N-acetylgalactosami1978 V: Oligosaccharides of human milk. I. From www.bloodjournal.org by guest on June 14, 2017. For personal use only. GENOTYPE Isolation IDENTIFICATION and OF characterization. ABO 123 SYSTEM Arch Biochem Biophys of ABO I 30:509, I 969 10. tives Barker R, Olsen of uridine cation Shaper and of l’etude lmm-h#{233}matol I 2. the blood Furuhata groups. I 3. Segfried JH, Hill RL: des J Biol Chem de antig#{233}nes de groupes sujets 19:105, T: Agarose N-acetylglucosamine B normaux 35, in a family 14. purifi- blood 1972 within weak B antigens. Vox Sang 9:268, Rev Fr 41:320, Trans 16. Am J Phys H, Walewska review Anthropol I, Boguslawa of the 13:109, gene-hypothesis of Okubo mode Y, Hazawa F: of inheritance. An Proc A2B3 Jpn phenotype Acad 41:316, inheritance Gent A note added to Yamaguchi et al. Proc Jpn Acad 1965 Van DeGrouchy group 1929 W: Unusual T: I 5. Komai A et B. ApplicaAB. H, atypical 1965 1976 A summarized Yamaguchi showing s#{233}riques associ#{233}es sanguins et Cis deriva- for the 247:71 J: Activit#{233}s glycosyltransf#{233}rasiques Ia biosynthese tion KW, diphosphate of galactosyltransferase. 1 1 . Badet a group I 964 Cong J, N, to chromosome 10:241, Weil D, Finaz Fr#{233}zalJ: Assignment 1976 9 in man-hamster C, Cochet of the C, Rebource ABO-Np-AK, hybrids. Cytogenet R, linkage Cell From www.bloodjournal.org by guest on June 14, 2017. For personal use only. 1980 55: 119-123 Identification of genotype of blood group A and B A Yoshida Updated information and services can be found at: http://www.bloodjournal.org/content/55/1/119.citation.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.