OPENSUBJECT AREAS:
EVOLUTIONARY
BIOLOGY

An integrative evolution theory ofhisto-blood group ABO and related
genes

GLYCOBIOLOGYIMMUNOGENETICS

Received
17 April 2014
Accepted
19 September 2014
Published
13 October 2014

Correspondence andrequests for materials
should be addressed toF.Y. (fyamamoto@imppc.org)

Fumiichiro Yamamoto1, Emili Cid1, Miyako Yamamoto1, Naruya Saitou2, Jaume Bertranpetit3& Antoine Blancher4
1

ABO Histo-blood Groups and Cancer Laboratory, Cancer Genetics and Epigenetics Program, Institut de Medicina Predictiva i
Personalitzada del Càncer (IMPPC), Campus Can Ruti, Badalona, Catalonia, Spain, 2Division of Population Genetics, National
Institute of Genetics, Mishima, Japan, 3IBE - Institute of Evolutionary Biology (UPF-CSIC), Universitat Pompeu Fabra, Barcelona,
Catalonia, Spain, 4Laboratoire d’Immunogénétique Moléculaire (LIMT, EA3034), Faculté de Médecine Purpan, Université Paul
Sabatier (Université de Toulouse III), Toulouse, France.

The ABO system is one of the most important blood group systems in transfusion/transplantation
medicine. However, the evolutionary significance of the ABO gene and its polymorphism remained
unknown. We took an integrative approach to gain insights into the significance of the evolutionary processof ABO genes, including those related not only phylogenetically but also functionally. We experimentallycreated a code table correlating amino acid sequence motifs of the ABO gene-encoded glycosyltransferases
with GalNAc (A)/galactose (B) specificity, and assigned A/B specificity to individual ABO genes fromvarious species thus going beyond the simple sequence comparison. Together with genome information andphylogenetic analyses, this assignment revealed early appearance of A and B gene sequences in evolution andpotentially non-allelic presence of both gene sequences in some animal species. We argue: Evolution may
have suppressed the establishment of two independent, functional A and B genes in most vertebrates andpromoted A/B conversion through amino acid substitutions and/or recombination; A/B allelism shouldhave existed in common ancestors of primates; and bacterial ABO genes evolved through horizontal andvertical gene transmission into 2 separate groups encoding glycosyltransferases with distinct sugar
specificities.
he human histo-blood group ABO system is crucial in safe blood transfusion and cell/tissue/organ transplantation1,2. This system consists in A and B oligosaccharide antigens expressed on red blood cells (RBCs)
as glycoproteins and glycolipids and antibodies against those antigens in serum. A and B antigens are also
expressed by epithelial and endothelial cells, and in secretor type individuals they are also expressed on mucins
secreted by exocrine glands. The immuno-dominant structures of A and B antigens are GalNAca1-.3(Fuca1.2)Gal- and Gala1-.3(Fuca1-.2)Gal-, respectively. A and B alleles of the ABO genetic locus encode A and B
transferases, which respectively transfer an N-acetyl-D-galactosamine (GalNAc) or a D-galactose (Gal) to H
substances with an a1,3-glycosidic linkage. H substances with the Fuca1-.2Gal- structure are synthesized byfucosylation catalyzed by a1,2-fucosyltransferases (a1,2-FTs) encoded by FUT1/FUT2/SEC1 genes. FUT1encoded a1,2-FTs and FUT2/SEC1-encoded a1,2-FTs exhibit distinct acceptor substrate specificity, and are
differentially expressed amongst tissues. In humans SEC1 is a pseudogene and FUT2 gene presents frequent nullalleles so that about 20% of individuals are incapable of expressing either H, A, or B antigens in secretions (nonsecretor type). In the absence of a1,2-FTs no H antigens are produced. Therefore, A/B transferases function only
when at least one active a1,2-FT is simultaneously present.
In 1990 we correlated the nucleotide sequences of A, B, and O allelic cDNAs and the expression of A and B
antigens, and elucidated the molecular genetic basis of human histo-blood group ABO system3,4. Four amino acidsubstitutions (Arg176Gly, Gly235Ser, Leu266Met, and Gly268Ala) discriminate A and B transferases. A single
nucleotide deletion (261delG) was found in O alleles. We later identified mutations in A/B subgroup alleles (A2,A3, Ax, and B3) and mutations in cis-AB and B(A) alleles specifying dual expression of A and B antigens5–7. Anothertype of O allele, which lacks 261delG but contains a Gly268Arg substitution, was found afterward8. ABO alleles
registered in the Blood Group Antigen Gene Mutation Database exceed 250, and ABO has become one of the moststudied human genetic loci for its polymorphism9.

T

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ABO genes exist not only in humans but also in many other vertebrate species although ABH antigen expression patterns may bedifferent. In addition to A and B transferases, there are additional
enzymes transferring a GalNAc/galactose by a1,3-glycosidic linkage:
a1,3-galactosyltransferase and isogloboside 3 synthase (both ofgalactose specificity), and Forssman glycolipid synthase (GalNAc
specificity). These enzymes catalyze the last synthetic steps of a1,3galactosyl epitope (Gala1-.3Gal 1-.4GlcNAc -), isogloboside 3(Gala1-.3Gal 1-.4Glc 1-Ceramide), and Forssman glycolipid
antigen (GalNAca1-.3GalNAc 1-.3Gala1-.4Gal 1-.4Glc 1Ceramide), respectively. It should be noted that these enzymes utilize
other acceptor substrates than H substances as the chemical structures of their reaction products indicate. Genes encoding these a1,3Gal(NAc) transferases (a1,3-Gal(NAc)Ts) (GGTA1, A3GALT2, and
GBGT1 genes, respectively) are paralogous to the ABO gene, and they
are evolutionarily related10–13. Although transferase activity remains
to be demonstrated for its encoded protein, another paralogous genetic locus, GLT6D1 (glycosyltransferase 6 domain containing 1), wasassociated to periodontitis susceptibility14. Based on the nucleotide
and deduced amino acid sequences of ABO and related genes, a birthand-death evolution model was proposed15,16. Several theories havebeen proposed on the evolution of the primate ABO polymorphism17–22. And the dynamics of the human ABO gene evolution havebeen extensively studied23,24. A brief summary of prior knowledge
about ABO evolution will be presented in each individual sub-sectionin the Results section. Indisputably, sequences, single nucleotide
polymorphisms (SNPs), and mutations are critical to investigate
gene evolution. However, the analyses based solely on sequences
are insufficient especially because of genetic recombination. To interpret gene evolution properly knowledge of the gene-encoded proteins is fundamental. What is the protein function, which portion(s)of the protein are important for that function, where is the protein
located, does the protein form multimers, how does the protein
interact with other molecules, etc., all provide valuable information.
Especially, in order to investigate the ABO gene evolution the understanding of the sugar specificity of A and B transferases is essential.
As in many other areas of genetic studies, functional assays are of
critical importance.
In the present work, we analyzed many homologous genes andsequences that had been identified in various species through genome sequencing efforts. In addition to the sequences, we also utilized
additional data and information available: gene structure to determine whether a gene is partial or complete; chromosomal organization to deduce duplication(s), deletion(s), inversion(s), andtranslocation(s) that have occurred; and information on A/B transferases and A/B oligosaccharides to obtain clues on functionality.
Data were interpreted with caution because of the incompletenessof genome sequence databases, wrong annotations, and differencesamong individuals within a species, and errors in genome assemblies.
Based on mostly relevant, but not entirely accurate, data, we havedelineated a potential scenario of the ABO gene evolution. Taking
advantage of our expertise, we also prepared several dozens of aminoacid substitution constructs of the human A transferase in an
expression vector by in vitro mutagenesis, determined their
GalNAc/galactose specificity, and generated a code table correlating
amino acid sequence motif with A/B specificity. Utilizing this table,
we decoded the A/B specificity of the ABO genes annotated from avariety of species, which in turn has allowed us to uniquely evaluate
several critical hypotheses on the evolution of the ABO and related
genes and their functional impact.

Results
Gene duplications and changes in substrate specificity of theencoded glycosyltransferases created ABO family of genes inanimals. All the a1,3-Gal(NAc)T genes in genome databases thatwere analyzed are listed in Fig. 1. Species were aligned based on their
SCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

evolutionary relationship (human at top and lamprey at bottom)25. A
phylogenetic tree was constructed for the 104 protein sequences thatare likely to encode functional a1,3-Gal(NAc)Ts, and is shown in
Fig. 2. GBGT1, A3GALT2, GGTA1, and GLT6D1 genes formed
separate clusters, whereas both A and B genes were clustered into asingle ABO gene cluster. Except that many nonfunctional genes areomitted, these results obtained from amino acid sequence analysis
coincided well with the nucleotide sequence-based Ensembl gene tree
ENSGT00400000022032 and a previous report15.
The genes neighboring those glycosyltransferase genes are conserved well in many species and the consensus organizations areshown in Table 1. There is a wide variation in the repertoire of thosegenes among different species, and the model of birth-and-deathevolution26 fits well with the a1,3-Gal(NAc)T family of genes aspreviously reported15. For instance, amphibian Xenopus tropicalis
frog has ABO genes but lacks any other a1,3-Gal(NAc)T genes
whereas all the bird species examined have GBGT1 but lack
A3GALT2, GGTA1, and GLT6D1 genes.
Emergence of a1,2-fucosyltransferase genes preceded A/B transferase gene appearance in amphibians. Phylogenetic analyses andtheir chromosomal locations were used to separate FUT1, FUT2, and
SEC1 genes, and they are shown in 3 different columns in Fig. 1. The
distributions of these genes suggest that FUT2 gene was the oldest
a1,2-FT gene. FUT1 gene later appeared from FUT2 lineage aftergene duplication followed by acquisition of novel expressional/
enzymatic characteristics. SEC1 gene emerged much later after
duplication of FUT2 gene and following divergence from it,
confirming the evolutionary theory previously proposed of a1,2FT family of genes27. The chromosomal region containing a1,2-FT
genes has remained stable in many species, and the consensus isshown in Table 1.
A/B antigen expression was previously reported in frog species28,29.
As shown in Fig. 1, neither FUT1/FUT2/SEC1 genes nor ABO genes
are present in fish genomes. Contrastingly, amphibian Xenopus tropicalis frog has 4 FUT2 gene sequences, several of which seem toencode active a1,2-FTs. This frog species also contains multiple
ABO gene sequences, including a few with possible functionality.
Chinese softshell turtle and many mammalian genomes also possess
potentially functional a1,2-FT and A/B transferase genes. Therefore,
it is logical to hypothesize that A/B antigen(s) appeared after the
separation of fish and amphibian lineages.
A code table was generated to correlate amino acid sequence motif
with A/B specificity. Progresses have been made in understanding
A/B transferases over the last decade. Among the 4 amino acidsubstitutions at codons 176, 235, 266, and 268 between the human
A and B transferases, the third and fourth substitutions were shown
to be crucial for different donor nucleotide-sugar substrate specificity
whereas the second is influential and the first is not so important4.
Our in vitro mutagenesis study30 and the determination of the threedimensional structures of A/B transferases by others31 confirmed thecritical roles of amino acids at codons 266 and 268.
In this study we prepared a library of 40 amino acid substitution
constructs of human A transferase, which contained any one ofpotential 20 amino acid residues at codon 266 in combination witheither glycine of A transferase or alanine of B transferase at codon
268. Furthermore, we also prepared additional constructs at codons
263–268 that contained deduced amino acids present in annotated
ABO and related a1,3-Gal(NAc)T genes in the genome databases but
were not represented in the library. DNA from those constructs wastransfected to HeLa cells expressing cell-surface H substances, andthe expression of A/B antigens was examined immunologically,using antibodies against blood group A/B antigens, respectively. A
code table was generated that correlates amino acid sequence motifs
and A/B specificity of the enzymes encoded by the various constructs(Table 2). The activity is shown semi-quantitatively in a 4-fold expo2

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Figure 1 | Species-dependent distribution of FUT1/FUT2/SEC1 a1,2-fucosyltransferase genes and ABO/GBGT1/A3GALT2/GGTA1/GLT6D1 a1,3Gal(NAc) transferase genes. This table shows the distribution of a1,2-FT genes and a1,3-Gal(NAc)T genes in a variety of organisms. Ensembl gene
identifiers are listed only with the meaningful digits, excluding 0 s on the left from their IDs. Genes were categorized into groups based on Ensembl genetrees, chromosomal locations, and our own analyses, and they are aligned in different columns and shown highlighted in different colors. Amino acidsequences corresponding to the codons 266–268 of human A/B transferases are also shown. The symbol ‘‘---’’ indicates the absence of sequence motif, and‘‘N/A’’ means not annotated in databases. A single column of ‘‘Pseudo/Ancient’’ was used to list two types of annotated gene sequences: The ABO
retropseudogene sequences that were originally derived from an intronless cDNA are highlighted in tan color (Pseudo) and the sequences that formed acluster next to the ABO gene in the phylogenetic analysis are highlighted in yellow (Ancient). The gene sequences that formed a cluster outside of the ABO/
GBGT1 genes are highlighted in orange, and they are shown separately in the ‘‘ABO/GBGT1 Ancient’’ column. The annotated genes may or may not befunctional, the latter of which may also be called as O genes or pseudogenes. Note that genome sequences were not complete for many species, andtherefore, errors may exist. In addition, there are numerous homologous sequences that have yet to be annotated and mapped on chromosomes.
Furthermore, polymorphism may also exist.
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99
22

A
PC F-GA
LA -G BG
PV F-G BG T1
EE A- BG T1
OA U-G GBG T1
NBG T
23
PS GBG T1 1 6
I-G
T1
9 6
BG
TGU
2
T1
-GB
MGA GT1
99-GBGT1
GALDAR4GBG
T1
GBGT
94 35
1
99 69
DAR3-GB99
96
G T1
DAR5-GBGT1
99
99
DAR2-GBGT1
99
95
98
DAR1-GBGT1
54
XET2-ABOBO
XET1-A ABO
99
PSI-ABO 6969
OAN ABO
99
U70
MM -ABO
45
O BO
G
G R-A
55
PT -ABO O
99
4 9
A -AB O
9J
9C U1 BA O
ML U3- -ABO
ML LU2 -AB
M U2
C
O

98
91
96

9
GT1
-GB T1
6
1
G
SHA
T
B
D-G -GBG 1
41
MO
T
10
O
BG
CH
1
47
S-G T1 GT
2
MU GBG B
28 6
GO- CA- GT1 T1
CP
B BG
E
G
R- A-G
TT FC

C

0.1

GGO-A3GALT2PPY-A3GALT2
MMU-A3G29
ALT2
CJA-A3
25
GALT2
OGA
-A3G
ALT2
21 7
OPR
A
3
M
1
G
9
PU 29
A LT
2
28
ML A3GAL
91
U-A
T2
11
P
3
2
GA
LA V A
61 5
LT 2
DN F-A -A3G
3
OGA ALTS D O A 3G L T 2 2
CP TO- R-A ALTO A3 3GA 2-A GA
3G L L T 2
AL T2
T2

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76

75

99

95

37
34

1 1
TA A
G GT 1-G -G TAO D G A1
N O G
R M A- GT 1
SH A-G TA 1
G G G TA
99
O
1
O- GG
34
ST LAF- GGTAOCP TA1
15
5
-GG
1
A
U
65 2
OC A-GGT
A1
5 CJ
76 4
GGT
MICGTA1
17
MLU-G TA1
15
BTA-GG
38
SSC-GGTA11912
ECA-GGTA1
10 FCA-GGTA1
MPU-G A1GT
AME-GGT
A1
CAF-G
GTA1

O
RN CU
RN O3 1-A
RN O2 -AB BO
RN O4 -AB O
PC O1 -AB O 85
-A O
A
BT
95
A-A -ABO BO
99
B
O
CA
F
15
78
MPU -ABO
-AB
O
FCA
18
-ABO
90

99

T2 2
A L LTGA
32- A A 3G A LTE
E T U S A 3G
M OT2
R N G A L A LT 2
99
T2
A3 3G
C- I2-A GAL T2
99
A
3
L
L ON
A
I4- 3GAON C1-A ALT2
99
GA 1-A3G
LT 2
TRU 1-A3GA
73L
O R LT 2
35
3GA
64
TNI-A
LT2
A3GA
96
TRU2- 1-A3GALT2
GMO
9 9 99
GMO2-A3GALT2
38
44
ORL2-A3GALT2
57
XMA-A3GALT295
GAC2-A3G99
ALT241
ONI1-A
3GALT2
O
9
NI39 084 9
AME A3GALT2
-GLT
M
4
P
2
99
6D 1
CA U-GLT
6D 1
F-G
55
B
L
EC TA-G T6D1
85
A-G LT6
ML
D99
U - LT6D 1
GL
1
T
M OC
US U- MU 6D1
G-G L SG T6 GL
TA D
T6
D1
1 1

67
63
72

Figure 2 | Evolution of a1,3-Gal(NAc) transferase family of genes. The MEGA5 software was used to analyze 104 amino acid sequences potentially
encoding intact ABO proteins. The amino acid sequences corresponding to codons 69–354 of the human A transferase were examined. 1,000 bootstrap
replications were computed. Branches leading to ABO, GBGT1, A3GALT2, GGTA1, and GLT6D1 genes are colored in yellow, grey, green, purple, andblue, respectively. The bootstrap frequencies are shown on the branching points. Fishes, amphibians, reptiles, and birds are marked with closed circles inred, purple, green, and dark blue whereas mammals are unmarked. The species code names correspond to the names shown in the ‘‘Ensembl Database’’column in Fig. 1. For instance, PTR for chimpanzee (Pan troglodytes) is obtainable by removing ENS and G from the database name (ENSPTRG).

nential scale with 51 highest and - none. The motifs observed in
ABO genes in natura are shown in bold type.
The control constructs exhibited the anticipated specificity:
AGG motif at codons 266–268 in pig A gene, LGG and MGA in
human A and B alleles, and GGA in mouse cis-AB gene for A, A, B,
and AB specificity, respectively. The results clearly demonstrated
that the amino acid residue at codon 266 is crucial to determine
the sugar specificity and activity of the encoded transferase. Some
constructs possessing glycine at codon 268 exhibited different specificity/activity from those possessing alanine, suggesting thatcodon 268 is also important. A tendency of preferential use ofgalactose over GalNAc was observed by the Gly268Ala substitution, possibly because increased size in side chain at that position
hinders larger GalNAc access whereas facilitating smaller galactose
SCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

access. Several constructs with the amino acid sequence motifs thatwere overlapped with our previous study30 exhibited the same
specificity/activity in spite of the differences in the A/B transferase
backbone.
In addition to the constructs expressing either A or B transferase
activity, several constructs exhibited both A and B transferase activities whereas several others showed none. For instance, human A
transferase constructs containing AAA, CGG, or SGG motif exhibited A specificity, whereas those with IGA, MAA, MGS, or QGC
exhibited B specificity. The constructs with MGG, SGA, TGA, or
AAS showed both A and B specificity whereas those with AAN,
TEA, or TGF showed neither. An unexpected finding was that glycine at codon 267 is not an absolute pre-requisite for A/B transferase
activity. We next applied the codes to uniquely assign potential A/B
4

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Table 1 | Consensus organization of genes surrounding a1,3-Gal(NAc) transferase and a1,2-fucosyltransferase genes
a1,3-Gal(NAc) transferase genes
ABO and GBGT1 genes
REXO4-., ,-C9ORF96, SURF4-., ,-SURF2, SURF1-., ,-RPL7A, MED22-., SURF6-., ABO-., LCN1-., OBP2B-., GBGT1-., RALGDS-., ,-CEL,,-GTF3C5, ,-GFI1B
A3GALT2 gene
Mammals,-ZSCAN20, PHC2-., A3GALT2-., ,-ZNF362, TRIM62-.
Fish,-FAM83E, EMP3-., ,-A3GALT2, ,-ZNF362, ,-TRIM62
GUCA1B-., MAPK8IP1-., A3GALT2-., LRP4-., ,-NELL1
GGTA1 and GLT6D1 genes
TTLL11-., ,-DAB2IP, GGTA1 (-1)-., GGTA1 (-2)-., GLT6D1 (-1)-., STOM-., ,-GSN
OBP2A-., PAEP-., ,-GLT6D1 (-2), LCN9-., ,-SOHLH1, KCNT1-.
a1,2-fucosyltransferase genes
FUT1/FUT2/SEC1 genes
SULT2B-., ,-FAM83E, SPACA4-., ,-RPL18, SPHK2-., ,-DBP, ,-CA11, ,-NTN5, SEC1-., FUT2-., ,-MAMSTR, ,-RASIP1, ,-IZUMO1, ,FUT1, FGF21-., ,-BCAT2, ,-HSD17B14, ,-PLEKHA4, PPP1R15A-., ,-TULP2, NUCB1-.
Chromosomal regions containing a1,3-Gal(NAc)T and a1,2-FT genes have remained stable in many species with the consensus organization shown. The arrows indicate the direction of transcription.

specificity of the annotated ABO genes and critically evaluated several hypotheses on the evolution of the ABO genes.
A and B gene sequences appeared early in evolution and arepotentially present in a non-allelic manner in some species. The
first evidence of genomes with multiple copies of ABO genesequences came from the Southern hybridization experiments
showing multiple bands of hybridization in dog, rabbit, and rat
genomic DNA using a human probe32. Later studies demonstrated
multiple genes in rat33. As shown in Fig. 1, additional species also

seem to possess multiple ABO gene sequences. They are Xenopus
tropicalis frog, Chinese softshell turtle, platypus, microbat, dog,
ferret, panda, horse, Kangaroo rat, rat, and rabbit species. Genes
flanking full/partial ABO genes are shown for each individual
species in Table 3, together with the amino acid sequences
corresponding to codons 266–268 of the human A/B transferases.
We applied Table 2 to decode A/B specificity of individual ABO
gene sequences annotated in various vertebrate species. It was foundthat several species not only contain multiple copies of ABO genesequences but also they may have both A-specific and B-specific gene

Table 2 | Specificity and activity of human A transferase expression constructs containing various amino acids at codons 263–268(I). G at codon 268(II). A at codon 268(III). Additional

CodonsA

B

A/B

CodonsA

B

A/B

CodonsA

B

A/B(266–268)

Activity

Activity

Specificity(266–268)

Activity

Activity

Specificity(266–268)

Activity

Activity

Specificity

AGG
CGG
DGG
EGG
FGG
GGG
HGG
IGG
KGG
LGG
MGG
NGG
PGG
QGG
RGG
SGG
TGG
VGG
WGG
YGG

11111
11111
11
1111
2
1111
2
1111
2
11111
1111
11111
11111
1111
2
1111
11111
1111
11
2

2
2
11
2
1111
2
1111
1111
2
2
1111
1
2
111
2
2
2
2
1
1111

A
A
AB
A
B
A
B
AB
2
A
AB
AB
A
AB
2
A
A
A
AB
B

AGA
CGA
DGA
EGA
FGA
GGA
HGA
IGA
KGA
LGA
MGA
NGA
PGA
QGA
RGA
SGA
TGA
VGA
WGA
YGA

AB
AB
B
B
B
AB
B
B
B
AB
B
AB
A
B
2
AB
AB
AB
B
B

AAA
AAN
AAS
MAA
MGP
MGS
QGC
SSE
TAS
TEA
TGC
TGF
TSE

11111
11
1111
111
2
111
2
1111
2
1111
1111
111
2
1111
2
1111
2
111
11111
1
2
1111
1111
11
1111
2
2
11111
2
2
1111
111
1111
111
1111
111
2
1111
2
11

11111
2
2
2
1111
111
2
11111
2
111
2
11111
2
11111
2
2
2
2
2
2
1111
2
2
2
2
2

(263–268)
AYVYGS2FYFTSE2
HYYMGG 1111
YYYAGG 11111
YYYMGG 11111
YYYTGS 11111
YYYTSE2
YYYTSG 11111

2
2
1111
2
111
2
2
2

A
2
AB
B
B
B
B
2
2
2
A
2
2

2
2
AB
A
AB
A
2
A

The left 2 sets show the results of a library of human A transferase expression constructs containing any of 20 potential amino acid residues at codon 266 with glycine of A transferase or alanine of B
transferase at codon 268. The right set shows the results of additional constructs that were not included in the library. The results of immunostaining with anti-A or anti-B antibodies were adjusted bytransfection efficiency using co-transfected GFP-positive cell percentages. The activity is shown in a semi-quantitative manner on a 4-fold exponential scale with 51 highest and 2 none. The letter size in A/B
Specificity reflects the activity strength whereas ‘‘2’’ indicates no activity. The constructs shown in bold type are mentioned in the text.

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sequences in their genomes. For instance, Xenopus tropicalis frog has
A gene sequences with AGG or TGC motif and B gene sequenceswith MAA motif. Other species identified are: Chinese softshell turtle(AAA for A and MGA for B), platypus (AGG for A, MGA for B, andLGA for AB), horse and rat (AGG for A and MGA for B), microbat(LGG for A and MGA for B), and rabbit (LGG for A and MGA and
IGA for B). These results suggest that functional differentiation
between A and B gene sequences appeared early in evolution, possibly just after the ABO gene emergence in amphibians.
As shown in Table 3, horse A and B gene sequences are closely
located in tandem on the same chromosome. Therefore, if horse
genome assembly is correct, those sequences may not be unigenic
alleles. Microbat A and B gene sequences have not yet been mappedon chromosomes, however, at least one A and one B gene sequencesof the three present in the genome were aligned side-by-side within asingle contig (ENSMLUG00000029891 with LGG and ENSMLUG00000026173 with MGA in Scaffold GL431842: 18,18626,341). Accordingly, they are not allelic, either. The rat genome in
the Ensembl database lists 4 ABO gene sequences: 1 A (AGG) and 3 Bs
(MGA). The surrounding chromosomal organization in Table 3
shows that those sequences are not alleles. Rat A and B gene sequences
located tandemly in a cis-manner contrast to mouse gene (GGA)
encoding a transferase with dual specificity (cis-AB enzyme)34.
However, heterogeneity seems to exist among different strains ofrats. The Ensembl genome is from the BN/SsNHsdMCW strain. In
addition to this strain, GenBank database also houses the genome
sequence from another strain, the BN/Sprague-Dawley strain(Rn_Celera). 1 A (AGG) and 2 B (MGA) gene sequences, rather than
1 A and 3 B, were mapped for this strain. In another strain, Wistar, 3
A and 1 B gene sequences were cloned although they have not been
mapped33. Different cloning results were obtained from inbred GOTW strain35 and the BDIX strain36, further complicating the understanding of rat ABO genes.
In spite of potential errors and problems that are frequently associated with the sequences and genome assemblies of polymorphic
genes and multi-gene families, the presence of multiple copies of
non-allelic A and B gene sequences in rat and other species cannot
be all attributed to bioinformatics mistakes. Even if sequence alignment all failed from the same caveats, the case still stands with rats atleast. Because three different A and one B gene sequences were cloned
from a single Wistar rat, they cannot be allelic at a single genetic
locus33,37.
Many of non-allelic ABO protein sequences were clustered within
species in phylogenetic analyses. Phylogenetic trees of ABO
proteins/peptides were constructed from species having more than
1 annotated ABO gene (Fig. 3a). For comparison, the human A and B
transferase sequences were included in the analysis although humansequences are allelic. Proteins corresponding to full genes withinitiation and termination codons are marked with circles, whereas
peptides corresponding to partial genes are marked with triangles.
The symbols’ colors indicate deduced potential A/B specificity(GalNAc, galactose, both, none, and uncharacterized specificity are
shown in red, green, yellow, blue, and black, respectively). The aminoacids corresponding to codons 266–268 of the human A transferase
are shown in parentheses.
The majority of ABO protein sequences were clustered in speciesspecific groups, including platypus, microbat, rabbit, and rat.
However, several protein sequences from two distant species areon a common phylogenetic branch. Among them, two frog (bothwith MAA motif) and two turtle (with AAN and AAS motifs)
sequences clustered together. However, those sequences werededuced to be nonfunctional, having aberrant gene organizations
such as the absence of N-terminal exons or missing initiation/termination codons. Two ferret (IGA or MEA) and three panda (MGP,
MGA, and ---) protein sequences corresponding partial genes withSCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

aberrations in codon reading frame and gene structure, clustered on acommon branch, apart from the ferret protein from a full gene with
AGG motif. In horse species two genes (MGA and AGG) that arelocated side-by-side on the same chromosome were separated in thephylogenetic tree, possibly due to frameshift mutations deleting aserine close to MGA motif (MGAFFGGSV) and the accelerated
accumulation of mutations after inactivation.
An intronless ABO gene cDNA was integrated into the mammalian genome. In addition to full/partial genes, ABO retropseudogenes also exist, originally derived from an intronless ABO gene
cDNA that was integrated into the genome during the mammalianevolution (Fig. 1). Those retropseudogenes clustered separately fromfull/partial ABO genes in phylogenetic analyses, and a phylogenetic
tree of ABO retropseudogene products is shown in Fig. 3b. This treesuggests that the original sequence may have contained a TGA motif,
which is present in some bacterial ABO genes (see below), but ismissing in animal ABO genes that were analyzed other than the
retropseudogenes. The implication and potential significance areunknown.
Several different molecular mechanisms may be responsible foranimal AO polymorphism. Generation of enzymes with novel
specificity and/or creation of genes with differential expression
patterns must suffice special conditions and requirements. On thecontrary, inactivation of gene function or annulment of transferase
activity may be relatively easily achieved. Diverse inactivation
mechanisms, including frameshift and missense mutations, havebeen identified in human O alleles4,8,16,23,38,39. Additionally, speciesspecific O alleles, which possibly resulted from independent silencing
mutations, are known to exist in non-human primates40–42. In nonprimate animal species unigenic AO polymorphism has beenreported of pig, dog, rat, cow, and rabbit43. The molecular mechanism of the porcine AO polymorphism was previously elucidated44,45.
A major portion of the structural gene, including the entire coding
sequence in the last coding exon, was found missing in O alleles fromvarious pig strains.
Assignment of A/B specificity to individual ABO gene sequences
has allowed us to investigate the molecular mechanisms that established AO polymorphism in other species. Two genes are annotatedin dog species (with AGG or SGG). The AGG sequence is located in
the consensus chromosomal region, but the SGG sequence is locatedon a different chromosome and seems to be nonfunctional as judged
by abnormal gene structure with the last coding exon indel-disrupted. Therefore, AO polymorphism is suspected at the AGG gene
locus. The examination of the coding sequence identified two interesting SNPs: rs9240920 [897G-.A] and rs9240927 [701delG]. The
former is a nonsense mutation (Trp299Ter) and the latter is a frameshift mutation. Therefore, the genes with either of these SNPs may
account for some of the O alleles in the dog AO polymorphism.
An interesting finding was made when the chromosomal organization surrounding the ABO genes was compared between rat andmouse species. The mouse genome is of very high quality, and many
duplicated regions have been properly solved. Therefore, it provides
a useful control. The gene organizations are similar except that a
DNA fragment containing 3 ABO (1 A and 2 B) and several additional genes is present in rat between ABO and FAM69B genes(Table 3). The genes present specifically in this chromosomal region
in the rat genome are shown in bold type. If the insertion occurred at
the population level, the genome without the insert may be regarded
as O allele. Alternatively, O alleles may have arisen from the genome
with A gene by deletion/unequal crossover. The cow and rabbit
genomes list one (A gene sequence with AGG motif) and four (1 A
gene sequence with LGG motif, 1 B gene sequence with IGA, and 2 B
gene sequences with MGA, in addition to 4 retropseudogene
sequences), respectively. The information on the ABO genes in those6

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Table 3 | Genes adjacent to ABO genes
Species

Gene order*

Primates
Human (Homo sapiens)
Chimpanzee (Pan troglodytes)
Gorilla (Gorilla gorilla gorilla)
Orangutan (Pongo abelii)
Rhesus macaque (Macaca mulatta)
Marmoset (Callithrix jacchus)
Bushbaby (Otolemur garnettii)
Other Mammals
Mouse (Mus musculus)
Rat (Rattus norvegicus)
Rabbit (Oryctolagus cuniculus)
Dog (Canis lupus familiaris)
Ferret (Mustela putorius furo)
Horse (Equus caballus)
Cow (Bos taurus)
Microbat (Myotis lucifugus)
Elephant (Loxodonta africana)Opossum (Monodelphis domestica)
Platypus (Ornithorhynchus anatinus)
Birds
Flycatcher (Ficedula albicollis)
Zebra finch (Taeniopygia guttata)
Turkey (Meleagris gallopavo)
Duck (Anas platyrhynchos)
Chicken (Gallus gallus)
Reptiles:Softshell turtle (Pelodiscus sinensis)
Amphibians:
Xenopus frog (Xenopus tropicalis)

1-., ,-2, ,-3, 4-., ,-6, 7-., 8-., ABO (LGG)-., 9-., 10-., GBGT1 (GGA)-., 11-., ,-12, ,-13, ,-14, ,-15
1-., ,-2, ,-3, ,-16, 4-., 7-., 8-., ABO (LGG)-., 10-., GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15
1-., ,-2, ,-3, 4-., ,-6, 7-., 8-., ABO (MGA)-., 9-., 10-., GBGT1 (GGA)-. 11-., 17-., ,-13, ,-14, ,-15
1-., ,-2, ,-3, 5-., 4-., ,-6, 7-., 8-., ,-18, 9-., 10-., GBGT1 (GGA)-., 11-., ,-19, 20-., ,-13, ,-14, ,-15
1-., ,-2, 5-., ,-3, 4-., ,-6, 7-., 8-., 21-., ABO (MGA)-., 9-., 10-., GBGT1 (GGK)-., 11-., ,-13, ,14, ,-15
1-., ,-2, 5-., ,-3, 4-., ,-ABO (LGG), ,-8, ,-7, 9-., 10-., GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15
1-., ,-2, ,-3, 4-., ,-6, 7-., 8-., ABO (LGG)-., ,-22, 23-., ,-24, 25-. // 26-., GBGT1 (GAA)-.
1-., ,-27, 5-., ,-3, 4-., ,-6, 7-., 8-., ABO (GGA)-., 28-., ,-22, 23-., ,-24, 25-. // 29-., ,-30, 31., GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15
1-., ,-32, 5-., ,-3, 4-., ,-33, 7-., 8-., ABO (MGA)-., 10-., ,-28, ,-ABO (AGG), 34-., 4-., ,-3,,-35, 7-., ABO (MGA)-., ABO (MGA)-., ,-10, 36-., ,-37, ,-38, ,-39, 40-., ,-22, 23-.,,-24, 25-.
ABO (LGG)-. // ABO (MGA)-. // ABO (MGA)-. // 42-., 43-., ABO (IGA)-., 44-., 45-.
5-., ,-3, ,-6, 7-., 8-., ABO (AGG)-., 9-., 46-., 46-., ,-GLT6D1 (DGS), 47-., 48-., 49-., ,-50, 51-.,,-52 // 29-., ,-53, ,-54, 31-., ,-55, GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15 // 56-., 57-., 58-.,
59-., ,-60, ,-61, 62-., 63-., ABO (SGG)-., 64-., ,-65
1-., ,-2, 5-., 4-., 7-., 8-., ,-ABO (AGG), GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15 // ,-66, 67-., ,68, 69-., 70-., ,-71, ABO (IGA)-., ,-72, 73-. // ,-74, 75-., 76-., ,-77, 78-., ,-79, ,-79, ABO(MEA)-., ,-80
1-., ,-2, ,-3, 4-., ,-6, 7-., 8-., ABO (AGG)-., ABO (MGA)-., GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15
1-., ,-2, ,-3, 4-., ,-6, 7-., 8-., ABO (AGG)-., ,-22, 23-., ,-24, 25-. // ,-51, 50-., ,-47, GLT6D1(DGA)-., ,-81, ,-46, ,-10, GBGT1 (GRA)-., 11-., ,-13, ,-14, ,-15
ABO (LGG)-., ,-ABO (MGA) // ,-ABO (MGA)
1-., ,-2, 4-., ,-6, 7-., 8-., ABO (AGG)-., 82-., ,-83 // ,-84, ,-13, ,-85, GBGT1 (GGA)-., 11-., ,14, ,-15,-3, ,-6, 4-., 7-., 8-., ,-ABO (MGG), 86-., GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15
ABO (AGG)-. // ABO (---)-., ABO (MGA)-. // ABO (LGA)-., 86-. // GBGT1 (GGA)-.
1-., ,-2, 5-., ,-3, 4-., ,-6, 7-., 8-., GBGT1 (GGA)-., 11-., ,-14, ,-13, ,-15 // 87-., 88-., 89-., ,ABO (TAS), 90-., 91-.
1-., ,-2, ,-2, 5-., ,-3, 4-., ,-6, 7-., 8-., 8-., GBGT1 (GGA) -., 11-., ,-14, ,-13, ,-15 // ,-92, ,-93,,-94, 95-., ,-96, ,-ABO (TAS), 97-., 98-.
1-., ,-2, 5-., ,-3, 4-., ,-6, 7-., 8-., 86-., GBGT1 (GGA)-., 11-., ,-14, ,-13, ,-15
1-., ,-2, 5-., ,-3, 4-., ,-6, 7-., 8-., 86-., GBGT1 (GGA)-., 11-., ,-14, ,-13, ,-15
1-., ,-2, 5-., ,-3, 4-., ,-6, 7-., 8-., 86-., GBGT1 (GGA)-., 11-., ,-14, ,-13, ,-15
99-., ,-100, ,-101, 1-., ,-2, ,-ABO (AAA), 5-., ,- 3, 4-., ,-6, 7-., 8-. // ABO (AAA)-. // ABO (AAA). // ABO (MGA)-. // 86-., GBGT1 (GGA)-., 11-., ,-13, ,-14, ,-15 // 102-., 103-., ,-ABO (AAN),
104-., 104-. // ,-ABO (AAS),-6, 7-., ,-105, 8-., ABO (AGG)-., ABO (AGG)-., ABO (AGG)-., ABO (AGG)-. // ABO (AGG)-., ABO(AGG)-., ABO (TGC)-., ABO (TGC)-., ABO (TGC)-., 86-. // ,-106, ,-107, ,-108, 109-., ,-110,
ABO (MAA)-., ,-41 // ABO (MAA)-.

When there is long gap, double slash (//) is given. Three key amino acid sequences are shown in parentheses for ABO, GBGT1, and GLT6D1 genes. The genes in the inserted chromosomal region that is
specifically present in the rat genome and is absent in the mouse genome are shown in bold type. Other genes are abbreviated as follows.
1: REXO4
7: MED22
13: CEL
19: ENSPPYG019722
25: NOTCH1
31: MRPS2
37: OBP2A
43: MTPN
49: ENSCAFG019747
55: EEF1A1
61: ENSCAFG029179
67: YIPF3
73: ZNF318
79: FRY
85: FXN
91: OR9K2
97: SDR39U1
103: OR14I1
109: ANGPTL1

2: C9ORF96
8: SURF6
14: GTF3C5
20: ENSPPYG019721
26: MUS81
32: RGD1307355
38: RPL9
44: ENSOCUG029177
50: SOHLH1
56: IFIT2
62: ZNF487
68: TJAP1
74: PDS5B
80: RXFP2
86: CCDC180
92: SOST
98: DAD1
104: OR11A1
110: RASAL2

3: SURF2
9: LCN1P1 5 LCN1
15: GFI1B
21: ENSMMUG032079
27: GM711
33: RGD1560194
39: VEGP1
45: ARHGAP20
51: KCNT1
57: IFIT3
63: ZNF33A
69: LRRC73
75: N4BP2L2
81: LGB
87: NDC80
93: DUSP3
99: SLC2A6
105: A4GNTSCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

4: SURF1
10: OBP2B
16: ENSPTRG039599
22: FAM69B
28: LCN4
34: GOT2
40: VEGP2
46: PAEP
52: CAMSAP1
58: IFIT1
64: ZNF37A
70: DLK2
76: N4BP2L1
82: INSL6
88: USP17L23
94: MPP3
100: CACFD11
106: TOR3A

5: SURF4
11: RALGDS
17: ENSGGOG027486
23: AGPAT2
29: PPP1R26
35: RGD1560194
41: FAM5B
47: LCN953: ENSCAFG032138
59: IFIT5
65: CHRM3
71: ABCC10
77: BRCA2
83: JAK2
89: OR10AG1
95: KCNJ3
101: ADAMTS13
107: FAM20B

6: RPL7A
12: CELP
18: ENSPPYG019727
24: EGFL7
30: C9ORF116
36: RPS13
42: TRIB1
48: ENSCAFG019749
54: ENSCAFG031986
60: ZNF248
66: POLR1C
72: SAP18
78: ZAR1L
84: TJP2
90: OR6Y1
96: ACR
102: OR5AP2
108: RALGPS2

7

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I8Y940 Bacteroides ovatus CL02T12C04 (AGG)
D4WAD3 Bacteroides ovatus SD CMC 3f (AGG)
A7LVT2 Bacteroides ovatus ATCC 8483-1 (AGG)
C3QVK4 Bacteroides sp. 2_2_4 (AGG)
D0TMW4 Bacteroides sp. 2_1_22 (AGG)
C3Q957 Bacteroides sp. D1 (AGG)
D4VHD4 Bacteroides xylanisolvens SD CC 1b (AGG)
D4WYC8 Bacteroides ovatus SD CC 2a (AGG)
K5ZTG7 Parabacteroides goldsteinii CL02T12C30 (AGG)
N2AM13 Parabacteroides sp. ASF519 (AGG)
F8KUR1 Parachlamydia acanthamoebae UV7 (AGG)
D1R6G6 Parachlamydia acanthamoebae H. coccus (AGG)
E4TIL1 Calditerrivibrio nitroreducens DSM19672 (CGG)
D1B3X0 Sulfurospirillum deleyianum ATCC 51133 (CGG)
D3UIY4 Helicobacter mustelae ATCC 43772 (YGS)
D1PRD4 Subdoligranulum variabile DSM 15176-2 (AGG)
D1PIY3 Subdoligranulum variabile DSM 15176-1 (CGG)
M5PWJ9 Desulfovibrio africanus PCS-1 (CGG)
M5PWK3 Desulfovibrio africanus PCS-2 (CGG)
N9YX63 Clostridium bolteae 90B8 (CGG)

ENSAMEP008279 Panda (MGP)
ENSAMEP001042 Panda (MGA)
ENSAMEP001554 Panda (---)
ENSMPUP012913 Ferret (IGA)
ENSMPUP001967 Ferret (MEA)ENSECAP012378 Horse (MGA)ENSMLUP018586 Microbat (LGG)
ENSMLUP022379 Microbat (MGA)ENSMLUP008669 Microbat (MGA)ENSDORP013980 Kangaroo rat (MGA)ENSDORP005699 Kangaroo rat (MGA)ENSP439154 Human A allele (LGG)
ENSP439154 Human B allele (MGA)ENSOCUP019695 Rabbit (IGA)ENSOCUP007476 Rabbit (LGG)
ENSOCUP014616 Rabbit (MGA)ENSOCUP025476 Rabbit (MGA)ENSMICP016321 Mouse Lemur (SGA)ENSMICP011111 Mouse Lemur (MGA)ENSRNOP039997 Rat (MGA)R0BXE6 Clostridium bolteae 90A9 (CGG)
N9ZL05 Clostridium bolteae 90B3 (CGG)
C6YVD3 Francisella philomiragia ATCC 25015 (MGG)
D3I9S3 Prevotella sp. oral taxon 299 F0039 (MGG)
A5ZC71 Bacteroides caccae ATCC 43185 (MGG)
A7M0P3 Bacteroides ovatus ATCC 8483-2 (MGG)
D1QTJ7 Prevotella oris F0302 (MGS)
D7NC57 Prevotella oris C735 (MGS)
C7XFE5 Parabacteroides sp. D13 (QGG)
D0TJP2 Bacteroides sp. 2_1_33B (QGG)
B0NSM3 Bacteroides stercoris ATCC 43183 (QGG)
K1FD74 Bacteroides fragilis HMW 616 (QGG)
L3JHW3 Escherichia coli KTE237 (QGC)
Q5JBG6 Escherichia coli-2 (QGC)
L3J5Q1 Escherichia coli KTE236 (QGC)
L4XQK6 Escherichia coli KTE125 (QGC)

ENSRNOP067875 Rat (MGA)ENSRNOP057988 Rat (MGA)ENSRNOP065701 Rat ( AGG)ENSMPUP012325 Ferret (AGG)
ENSCAFP033395 Dog (AGG)
ENSCAFP032845 Dog (SGG)
ENSECAP017593 Horse (AGG)
ENSOANP016919 Platypus (MGA)ENSOANP019342 Platypus (LGA)ENSOANP023992 Platypus (AGG)
ENSOANP000558 Platypus (---)
ENSPSIP010227 C. softshell turtle (MGA)
ENSPSIP011627 C. softshell turtle (AAA)ENSPSIP009927 C. softshell turtle (AAA)ENSPSIP018376 C. softshell turtle (AAA)ENSPSIP005790 C. softshell turtle (AAN)

E1I6K1 Escherichia coli MS 78-1 (QGC)
I2R1M9 Escherichia sp. 4_1_40B (QGC)
L3VT57 Escherichia coli KTE161 (QGC)
L4PIQ3 Escherichia coli KTE202 (QGC)
Q58YW2 Escherichia coli-1 (QGC)
B0UVK9 Haemophilus somnus 2336 (MGS)
Q0I1L8 Haemophilus somnus 129Pt (MGS)
J8T7Q1 Pasteurella multocida P52VAC (MGS)J0MZK3 Capnocytophaga sp. oral 335 F0486 (MGS)
E4MU68 Capnocytophaga ochracea F0287 (MGG)
J0X995 Capnocytophaga ochracea Holt 25 (MGG)
N9EWC6 Acinetobacter calcoaceticus DSM30006 (TGA)
N8NA26 Acinetobacter calcoaceticus NIPH 13 (TGA)
D0S6I6 Acinetobacter calcoaceticus RUH2202 (TGA)
N9LP58 Acinetobacter sp. NIPH 284 (MGG)
G4CKK7 Neisseria shayeganii 871 (TGA)
N8Q1P4 Acinetobacter sp. CIP A162 (TGA)
D0SG80 Acinetobacter johnsonii SH046 (TGA)
A5WE44 Psychrobacter sp. PRwf-1 (TGA)
F9Q7G3 Haemophilus pittmaniae HK 85 (TCA)ENSPSIP004886 C. softshell turtle (AAS)ENSXETP023435 Xenopus frog (MAA)ENSXETP049370 Xenopus frog (MAA)ENSXETP010967 Xenopus frog (AGG)ENSXETP063821 Xenopus frog (AGG)ENSXETP010979 Xenopus frog (AGG)ENSXETP010980 Xenopus frog (AGG)ENSXETP032771 Xenopus frog (AGG)ENSXETP063088 Xenopus frog (AGG)ENSXETP063337 Xenopus frog (TGC)ENSXETP010992 Xenopus frog (TGC)ENSXETP010993 Xenopus frog (TGC)

0.20

0.10

0.00

b)

ENSCAFP032964 Dog (AGA)ENSCAFP010717 Dog (IGA)
ENSAMEP020264 Panda (IGA)ENSECAP003408 Horse (TGA)ENSECAP003484 Horse (TGA)ENSMMUP005099 Macaque (TGA)A G e nes
es

0.8

0.6

0.4

0.2

B /AB
G e neses

0.0

ENSOCUP017123 Rabbit (TEA)ENSOCUP025592 Rabbit (TEA)ENSOPRP003341 Pika (TGA)ENSOCUP018866 Rabbit (IGA)ENSOCUP023219 Rabbit (IGA)
ENSOGAP021391 Bushbaby (TGA)
ENSOGAP017532 Bushbaby (TGA)ENSLAFP020677 Elephant (TGF)

0.30

0.20

0.10

0.00

Figure 3 | (a): A phylogenetic tree of ABO proteins/peptides from species possessing multiple copies of ABO gene. Phylogenetic analyses were performed
with protein/peptide sequences from species that contain more than one ABO genes in their genomes. Processed intronless retropseudogenes were
excluded from analysis. The amino acid sequences were analyzed in its entirety. Potentially functional proteins from full genes with the initiation andtermination codons and peptides from partial genes without them are marked with circles and triangles, respectively. The symbol’s color indicatespotential sugar specificity (GalNAc, galactose, GalNAc/galactose, none, and unknown for red, green, yellow, blue, and black, respectively). Amino acidsequences corresponding to the codons 266–268 of human A/B transferases are also shown in parentheses. Genes in the same species are bracketed. Whenpotential A and B gene sequences are both present in a single species, the bracket was colored in red. Horse genes and ferret genes in 2 separate clusters arebracketed in blue and purple, respectively. Other species are bracketed in dark blue. (b): A phylogenetic tree of originally intronless ABO retropseudogene
products. The entire protein sequences of processed retropseudogenes were analyzed. Branches leading to different amino acid sequences at the important
positions are coded in different colors. (c): ABO gene evolution in bacteria. EMBL-EBI InterPro database listed 57 bacterial proteins within the GT6
family. 56 proteins/peptides, excluding 1 short one, were aligned to construct a phylogenetic tree. A gene from Helicobacter mustelae and B gene from
Escherichia coli O86 strain were included in the study, and their results are shown in bold type. The B gene-encoded protein (E1I6K1) consists of 234
amino acids, and the bacterial protein sequences corresponding to codons 2–219 of this protein were analyzed. The amino acid sequence motifs
corresponding to the codons 266–268 of human A/B transferases are also shown in parentheses. In E1I6K1 these correspond to codons 145–147. The
symbols’ color indicates sugar specificity of transferases: red, green, and yellow for GalNAc, galactose, and both, respectively, assuming that they arefunctional.
SCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

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species is currently fragmental, and their inactivating mechanisms of
O alleles remain to be determined.
A/B allelism should have existed in primate ancestors, and later
inactivation at population level resulted in ABO polymorphism.
Several primates exhibit ABO polymorphism, and the repertoire oftypes are species-dependent40. The inter-species sharing of the ABOpolymorphism led Landsteiner and Wiener to conceive the theory oftrans-species evolution of polymorphism. In this concept the allele
coalescence time of the most recent common allele ancestor predates
the speciation time. We previously determined partial nucleotide
sequences of the ABO genes from several primate species and demonstrated that amino acid residues corresponding to codons 266
and 268 of human A/B transferases are conserved in all the species
examined, depending on A or B allele32. Later evolutionary analyses
led to the hypotheses of trans-species inheritance17,22, convergent
gene evolution18–20, and a combination of those21. Because the ABO
gene inheritance in primates was still controversial46, we re-visitedthe topic for further evaluation, with additional experimental data on
sugar specificity and activity of A/B transferases summarized in thecode table.
Genome sequences in databases do not cover ABO polymorphism.
Human reference and non-reference genes (both with LGG motif) in
Ensemble database represent O and A alleles, respectively. The chimpanzee, gorilla, and macaque genes with LGG, MGA, and MGA,
respectively, represent A, B, and B alleles from those species. In all
the primate species the chromosomal region containing ABO gene issimilar to the consensus with minor differences (Table 3). The current EMBL-EBI InterPro database hosts non-overlapping 65 ABO
protein/peptide sequences, including several proteins with MGG,
MGS, or LGA motif.
The phylogenetic trees of primate ABO genes are complex22.
However, A and B specificity may be ascribed to amino acid residues
corresponding to human codons 266 and 268 and their neighbors, bynarrowing down the scanning window. In this investigation we,
instead, evaluated the convergent evolution theory from an enzymological point of view. As shown in Table 2, the A to B conversion ofsugar specificity may be achieved not only by the change from LGG
to MGA motif, but also by other amino acid substitutions and evenwith single amino acid substitutions. Note that only one base change
may be sufficient for the conversion to FGG, HGG, or YGG motif
with B specificity. The B to A conversion is also possible by changing
to other amino acids than LGG. However, the conversion from MGA
to an A specific motif may need at least 2 nucleotide changes, even forthe single amino acid substitution to PGA.
Therefore, it is difficult to assume that the same LGG ,-. MGA
conversion occurred in so many different occasions during the evolution period of primates. Selection after random mutation(s) does not
explain the convergent evolution hypothesis because other motifs
than LGG and MGA are also enzymatically functional (seeTable 2). Rather, current distribution may be easily explained byassuming that functional A and B alleles were both present in thecommon ancestors of primates.
Bacterial ABO genes evolved into 2 separate groups with different
sugar specificities through horizontal and vertical gene transmission. In addition to eukaryotes, ABO specificity also exists inprokaryotes, especially in Gram-negative bacteria, which constitutethe bulk of intestinal flora47. The first two ABO genes cloned frombacteria are from O86 strain of Escherichia coli and from Helicobacter
mustelae, which express B and A antigens, respectively48,49. Analyzing
19 bacterial genes, horizontal gene transfer between eukaryotes andprokaryotes and among bacteria was proposed to explain the absenceof ABO genes in many species of invertebrates, plants, and fungi50.
Because recent microorganism genome sequencings have identified
additional bacterial ABO genes, we analyzed 56 bacterial proteins in
EMBL-EBI InterPro database, and constructed phylogenetic trees of
SCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

bacterial ABO genes. A tree is shown in Fig. 3c. In contrast tovertebrate ABO genes, all the bacterial A genes with GalNAc
specificity segregated from the B or AB genes with galactose or
GalNAc/galactose specificity, respectively. Another important
finding is that the bacterial ABO genes have a different variation in
the amino acid sequence motif from the animal genes. AGG and
CGG motifs were found in the A gene sequences, MGS and QGC
in the B gene sequences, and MGG, QGG, and TGA were in the AB
gene sequences. Whereas many of the motifs found in the bacterial ABO genes are also present in animal genes, QGG motif
seems to be unique to bacteria. TGA motif was found in animal
ABO retropseudogenes as described above. In Bacteroides andParabacteroides species ABO genes were clustered separately forpossible A gene sequences with AGG and possible AB genesequences with QGG or MGG. In other bacterial species theirgenes were grouped in either of the two big clusters of A or B/AB
genes.

Discussion
What is the evolutionary significance of the ABO gene and its polymorphism? We tackled this question, employing an integrative
approach with standard phylogenetic techniques combined withmolecular enzymology. Based on gene distribution, we first concluded that A/B transferase gene appeared after the separation offish and amphibian lineages. Requirement of A/B transferases foran a1,2-linked fucosylated substrate strongly supports preceding
emergence of a1,2-FT genes over A/B transferase genes. In this context it is noteworthy that coelacanth has a FUT2 gene sequence(although its functionality is questionable) and no ABO genesequence. However, because coelacanth genome sequence is preliminary, a possibility remains that ABO gene may also exist in coelacanth. If this happens to be true, A/B gene appearance may be dated
back to the time of lobe-finned fish appearance.
We created a code table correlating amino acid sequence motif
with A/B specificity (Table 2). However, it should be noticed thathaving an active enzyme motif does not guarantee the gene function
and sugar specificity. Mutation(s) in other position(s) may spoil the
enzymatic activity51. Care must be taken to interpret the results
because sugar specificity is based on the assumption that genesequences encode functional glycosyltransferases, which is notalways the case. A and B gene sequences can be O, depending on
their functionality context42. Moreover, the table reveals one discordance, concerning the AYVYGS motif. The human A transferase construct containing this motif (in place of FYYLGG) at codons 263–268
did not exhibit A transferase activity whereas the H. mustelae bacterial gene having this sequence was reported to exhibit A activity.
We assume that structural differences in other portions of the bacterial enzyme may have compensated for the activity variation.
We identified multiple copies of ABO gene sequences in a varietyof species (Fig. 1), some of which possess sequence(s) with A-specificmotif(s) and sequence(s) with B-specific motif(s) (Table 3). If multiple copies are found only in one species, the possibility exists thatthey were erroneously assembled. However, because this wasobserved in several different species, it seems unlikely that all thosefindings may be artifacts. In case of rats ABO gene duplication seems
undeniably proved33,37. The number of species having both A and B
gene sequences is expected to increase as new genome sequencing
projects proceed, providing that duplicated regions are properly
solved, which may be somewhat difficult in most NextGen sequencing projects. Irrespective of A/B specificity, phylogenetic analyses
clustered those ABO gene sequences into a single cluster that was
separated from the clusters of other a1,3-Gal(NAc)T genes (GBGT1,
A3GALT2, GGTA1, and GLT6D1) (Fig. 2).
It is evident that animal A and B genes did not evolve into two
separate genetic entities. Apparently, evolution suppressed the establishment of independent, functional A and B genes by certain
9www.nature.com/scientificreports
mechanism(s). However, proximity in genetic distance does notseem to be responsible for this failed separation in spite of the factthat A and B gene sequences are situated very closely on a chromosome in some species. GGTA1(-1), GGTA1(-2), and GLT6D1(-1)
genes are also closely linked, as well as SEC1 and FUT2 genes(Table 1). These genes, however, took independent evolution paths,
as opposed to A and B gene sequences which did not. As shown in
Fig. 1, the majority of GBGT1, A3GALT2, and GGTA1 genes possess
conserved motifs of GGA, HAA, and HAA, respectively. This restriction strongly suggests that those motifs are vital to their glycosylation
reactions. However, there are some variations in the motif with ABO
gene and more with GLT6D1 gene. A and B genes encode glycosyltransferases with distinct sugar specificity. However, both A and B
transferases utilize the same H substances. Although this sharing of
acceptor substrates may have contributed to mutual dependence of
those two genes to a certain degree, it is not sufficient because SEC1
and FUT2 genes encoding a1,2-FTs with similar enzymatic characteristics still formed separate phylogenetic clusters.
Two modes of appearance and inheritance of A and B genesequences in a given animal species may be contemplated to explain
the results in Fig. 3a. One is that those sequences with different sugar
specificity appeared recurrently after the separation from other analyzed species by convergent mutations. Another much likely possibility is that those sequences may have attained species-specificsequence homology through intergenic exchanges after A/B specificity was inherited from common ancestral genes. An examination ofgene organization revealed that full genes with initiation and termination codons are rare in those species possessing multiple ABO
gene copies. Many are partial genes that are incapable of encoding
functional glycosyltransferases by themselves. We speculate that they
may serve as a reservoir for genetic diversity to switch A/B specificity
through gene conversion, exon shuffling, or recombination. In several species multiple ABO gene sequences are closely linked to one
another, which facilitates recombination/gene conversion without
genetic catastrophe, producing new possible adaptations at a higher
rate than by nucleotide substitutions.
As mentioned above with rats, insertion/deletion/unequal crossovers/gene conversion seems to have occurred frequently at the ABO
gene locus. It may have reduced gene number from several to one on
certain occasions. Therefore, it is not too far-fetched to hypothesizethat differential deletions/crossovers may have resulted in differential outcomes. Starting from tandemly linked A and B genesequences, A and B alleles may have been created (the multigenicto-unigenic transition hypothesis). New functional allele(s) may
have been generated within partial and nonfunctional sequence(s)so far as changes in gene organization could restore their functionality to encode active enzymes that are expressed after being inserted
or copied in the functional gene(s). An example of such restored
function (and not merely changing it) has recently been demonstrated of human A allele by recombination from functional B allele
and nonfunctional O allele52. Those events may have taken place
before simians appeared. Rats and rabbits have A genes with AGG
and LGG, respectively. Therefore, prosimians and simians may haveinherited an A gene with LGG similar to Lagomorpha genes, rather
than Rodentia genes, because no genes with AGG motif are found inprimates22. An alternative explanation would be the unigenic-tomultigenic transition hypothesis: A/B allelism appeared first andthen natural selection favored duplication events in many species
to separate both alleles whereas this separation did not occur inprimates. This is an interesting hypothesis because it may easily
explain the absence of separate evolution of A and B genes.
However, it seems to be less likely because all the other species than
primates, which are known to have unigenic polymorphism, exhibit
AO, and not AB, polymorphism43.
Based on the relationship between amino acid motifs and A/B
specificity, we have shown that A and B alleles with LGG and
SCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

MGA motifs, respectively, existed in common ancestors of primates.
This suggests that they were inherited, most probably, in a transspecies manner. However, the fact that other motifs than LGG and
MGA also exist in some primate species signifies that mutations/recombination also happened, of which several may be the resultof convergent evolution. For instance, LGA motif is found in
Ecuadorian squirrel monkeys and humans, and MGG is found in
Ecuadorian squirrel monkeys, Weeper capuchins, and humans,
although cases of cis-AB (with LGA or MGG motif) are rare in
humans. These motifs may be derived from either LGG or MGA
by point mutation or by recombination of those two alleles, still
supporting the inheritance of an ancestral polymorphism with A
allele (LGG) and B allele (MGA) as prototypic alleles. MGS motif
in titi monkeys may have resulted from MGA by a single nucleotide
substitution, rather than from LGG by 2 amino acid substitutions.
In addition to primates, many other animal species analyzed also
maintain the prevailing motifs of LGG and MGA although AGG isalso frequent in non-primate animals. Considering that additional
motifs may also render the ABO gene-encoded proteins enzymatically active as demonstrated in the code table, those 3 motifs may beconsidered ancestral for those species. However, to evaluate thispossibility further characterization of additional ABO genes frommany other species, including amphibians and reptiles, will beneeded. ABO genes seem to have evolved under more or less constantselective pressure for some polymorphism in their catalytic specificity, which in some species is achieved by carrying different genecopies (multigenic polymorphism) and in some other species
through allelic polymorphism of a single gene (unigenic polymorphism). Whether the latter is limited to primate species or not needs tobe determined in order to conclusively prove or disprove the multigenic-to-unigenic transition hypothesis.
The A/B antigen expression depends on the A/B genotype of individual. Although human and several other species express A/B antigens on red blood cells, the expression on RBCs is relatively rare. On
the contrary, epithelial cells, including those of the gastrointestinal
tract, express A/B antigens in many species. Accordingly, its significance may be better found in that cell-type. Many of cell-surface
oligosaccharide structures are involved in microbial interactions,and ABH antigens are not an exception53. Actually, ABO polymorphism has been associated with certain infectious diseases54–56. The
presence/absence of A/B antigens and concordant absence/presenceof anti-A/B antibodies provide strong defensive lines against infection. Having ABO gene should be beneficial because many vertebrate
species maintain this gene. However, having both functional A and B
genes ubiquitously within species might not be so advantageous
because they may eventually lose anti-A/B antibodies. Rather, frequent gene conversion of A/B specificity producing amino acid substitutions or recombination with nonfunctional partial genes may
have conferred an adaptation against microbial attacks. Different
ABO phenotypes in different species and ABO polymorphism within
species may inhibit inter-species and intra-species infections,
respectively. Our results conformed to the hypothesis that hostorganisms attained the variation utilizing those two molecularmechanisms.
We unexpectedly observed the separate clustering of bacterial
ABO genes into 2 groups with different sugar specificities (A and
B/AB genes) (Fig. 3c), as opposed to animal ABO genes, of which A
and B genes did not evolve independently. Widespread presence of
A/B genes in bacteria47 indicates that ABO mimicry is advantageous
to survival. The bacterial ABO genes have been transmitted horizontally to different bacteria and vertically through generations. We reason that these mixed modes of gene inheritance have allowed thesegregated evolution of the bacterial ABO genes in 2 groups. It isevident that horizontal gene transfer has been providing bacteriawith easier adaptation against host defense system. Contrastingly,interactions with infectious agents may have stimulated the host10

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ABO gene evolution, as intra-species polymorphism may help thesurvival of host species by changing allele frequency through balancing selection.
In conclusion, the systematic functional analysis correlating
amino acid sequence motifs with A/B specificities opened a newvenue to investigate the ABO gene and protein evolution. Together
with phylogenetic analyses, we have gained invaluable insights into
the evolutionary significance of the ABO gene and its polymorphismand successfully decoded several important questions.

Methods
Materials. Reagents for PCR, restriction endonucleases, T4 DNA ligase, and otherenzymes were purchased from LifeTechnologies (Carlsbad, CA) and New England
BioLabs (Ipswich, MA). HeLa cells, human cancer cells of uterus, were originallyobtained from American Type Culture Collection (ATCC), and have been
maintained in the laboratory over a decade. Cell culture media, frozen
transformation-competent E. coli bacteria, and Lipofectamine 2000 were alsopurchased from LifeTechnologies. Oligodeoxynucleotides were custom-synthesized
at the same company. Anti-A and anti-B murine monoclonal antibody mixtures werefrom OrthoDiagnostic Systems (Piscataway, NJ), and Vectastain ABC System andDAB (3, 39-diaminobenzidine) substrate for color development were from Vector
Laboratories (Burlingame, CA).
In vitro mutagenesis of human A transferase expression construct. We employed a
PCR-mediated in vitro mutagenesis approach as previously described30. Degenerateoligodeoxynucleotides were used to introduce amino acid substitutions at codon 266
and 268 of human A transferase. The primers originally used for a library
construction were the followings:
FYV7 (T7-F): 59-TAATACGACTCACTATAGGG
FYV1 (SV40 polyA-R): 59-GAAATTTGTGATGCTATTGC
IMPPC235 (F): GGCGATTTCTACTACNNNGGGGSGTTCTTCGGGGGGTCIMPPC236 (R): GACCCCCCGAAGAACSCCCCNNNGTAGTAGAAATCGCC
The capitalized underlined letters N and S denote a mixture of 4 nucleotides (G/A/
T/C) and 2 nucleotides (G/C) at those positions. Human A transferase expression
construct57 prepared in pSG-5 vector (Stratagene, La Jolla, CA) was used as a PCR
template. Two consecutive rounds of PCR reactions were performed, first with FYV7
(T7-F) and IMPPC236 (R) primers and separately with IMPPC235 (F) and FYV1
(SV40 polyA-R) primers, and second by mixing both the reactions. The PCR products
were cleaved with SacII and BamHI restriction enzymes, and ligated with the SacIIBamHI vector fragment of human A transferase expression construct. After DNA
transformation of E. coli bacteria, plasmid DNA was prepared from transformant
colonies, sequenced, and the constructs containing intended amino acid substitutions
but lacking additional non-synonymous mutations were selected for DNA transfection experiments. For those constructs, which we failed to obtain by using degenerate
oliogodeoxynucleotide primers, and those constructs, which were not covered by thelibrary approach, specific primers were designed for individual constructions (notshown).
DNA transfection and immunostaining. HeLa cells were used as a recipient of DNA
transfection. These cells were derived from a type O individual and exhibit cell surface
H substances. When functional A/B transferases are expressed by DNA transfection,
H substances are converted to A/B antigens. We have used this system at various
occasions to examine the specificity and activity of A/B transferase variants30,57,58.
DNA transfection experiments were performed using 96-well plates as previously
described59. Lipofectamine 2000 reagent was used, following the manufacturer’s
instructions. DNA from the FUT2 expression construct prepared in pSG-5 and DNA
from the pEGFP-N1 vector (GenBank Accession #U55762) were co-transfected: theformer to increase the acceptor substrate availability and the latter to calculate thetransfection efficiency for activity adjustment. Two days after DNA transfection,
GFP-positive cells were counted. The next day, cells were fixed withparaformaldehyde and washed with PBS. After drying, cells were treated first witheither anti-A or anti-B monoclonal antibodies, second with biotinylated anti-mouse
IgM, then with Avidin/Biotinylated Peroxidase Complex (ABC), followed by color
development using DAB substrate. Stained cells were counted microscopically, andA/B specificity and activity were determined after adjusting the transfection efficiency
using GFP-positive cell counts. Because of variable detachment of cells from dishsubstratum during fixation and immunostaining procedures, data were presented in asemi-quantitative manner.
Databases, sequence alignment, and construction of phylogenetic trees. Nucleotide
and amino acid sequences, exon-intron organizations, and chromosomal locations of
a1,2-FT genes (FUT1/FUT2/SEC1) and a1,3-Gal(NAc)T genes (ABO/GBGT1/
A3GALT2/GGTA1/GLT6D1) were retrieved from Ensembl (www.ensembl.org/index.
html) and GenBank (www.ncbi.nlm.nih.gov/genbank/) genome sequence databases.
Protein/peptide sequences of the ABO genes were retrieved from the EMBL-EBIInterPro database (www.ebi.ac.uk/interpro/).
Ensembl genome sequence database (release 73) listed 89 annotated a1,2-FT genes
with 66 speciation nodes and 15 duplications in the ENSGT00390000001450 genetree and 255 annotated a1,3-Gal(NAc)T genes with 185 speciation nodes and 65

SCIENTIFIC REPORTS | 4 : 6601 | DOI: 10.1038/srep06601

duplications in the ENSGT00400000022032 gene tree. The phylogenetic tree in Fig. 2
was constructed by the neighbor-joining method60. JTT model61 was used for
estimating number of amino acid substitutions and 1,000 bootstrap replications werecomputed by using MEGA562. The phylogenetic trees in Fig. 3 were constructed by
Maximum Likelihood method, using the same software.
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Acknowledgments
This work was supported by the Instituto de Salud Carlos III (grant number PI11/00454)
from the Spanish Government and the fund from Institut de Medicina Predictiva i
Personalitzada del Càncer (IMPPC) to FY. We thank Mrs. Masako Mizuguchi forphylogenetic construction of Fig. 2 and Ms. Patricia Barrero for technical assistance.

Author contributions
F.Y. conceived the project. M.Y., E.C. and F.Y. prepared amino acid substitution constructsof the human A transferase, performed DNA transfection experiments, andimmunologically determined the A/B specificity of the individual constructs. F.Y. retrieved
sequence data and other information from databases, F.Y. and N.S. prepared phylogenetic
trees, and F.Y., N.S., J.B. and A.B. analyzed and interpreted results. F.Y. wrote themanuscript draft, and all the other authors participated in revision and editing.

Additional information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Yamamoto, F. et al. An integrative evolution theory of histo-bloodgroup ABO and related genes. Sci. Rep. 4, 6601; DOI:10.1038/srep06601 (2014).
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