THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 17, Issue of April 27, pp. 13701–13708, 2001
Printed in U.S.A.

Murine Equivalent of the Human Histo-blood Group ABO Gene Is acis-AB Gene and Encodes a Glycosyltransferase with Both A and B
Transferase Activity*
Received for publication, November 30, 2000, and in revised form, January 12, 2001
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M010805200

Miyako Yamamoto‡, Xiao-Hong Lin‡, Yoshihiko Kominato§, Yukiko Hata§, Reiko Noda¶,
Naruya Saitou¶, and Fumiichiro Yamamoto‡储From ‡The Burnham Institute, La Jolla Cancer Research Center, La Jolla, California 92037, the §Department ofLegal Medicine, Faculty of Medicine, Toyama Medical & Pharmaceutical University, Toyama 930-01, Japan, and the¶Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima 411, Japan

Histo-blood group A/B antigens are clinically important antigens in blood transfusion and organ transplantation. These
antigens are oligosaccharide antigens whose immunodominant
structures are defined as GalNAc ␣133 (Fuc ␣132) Gal- andGal ␣133 (Fuc ␣132) Gal- for A and B antigen, respectively.
Functional alleles at the ABO locus encode enzymes that catalyze the final step of synthesis. A alleles encode for A transferase, which transfers the GalNAc residues from the UDPGalNAc nucleotide-sugar to the galactose residue of theacceptor H substrates defined by Fuc ␣132 Gal-. B alleles
encode for B transferase that transfers the galactose residue
from UDP-galactose to the same H substrates. O alleles are
nonfunctional, null alleles. During the past decade, we havebeen studying the molecular genetic basis of the histo-bloodgroup ABO system (1). From a human gastric carcinoma cellline cDNA library, we were able to clone human A transferase
cDNA (2) based on the partial amino acid sequence of thesoluble form of A transferase purified from human lung (3).
Using cross-hybridization with A transferase cDNA probes, wethen cloned B transferase cDNA and nonfunctional O alleliccDNA from cDNA libraries made with RNA from colon adeno* This study was supported in part by funds from The Burnham
Institute, the Department of Defense Breast Cancer Research Program(DAMD 17-98-1-8168), and from Kyowa Hakko Kogyo Co. Ltd. The costsof publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.
The nucleotide sequence reported in this paper has been submitted to
the DDBJ/GenBankTM/EBI Data Bank with accession numbers
AB041038 and AB041039.
储 To whom correspondence should be addressed: The Burnham Institute, La Jolla Cancer Research Center, 10901 N. Torrey Pines Rd., La
Jolla, CA 92037. Tel.: 858-646-3116; Fax: 858-646-3173; E-mail:fyamamoto@burnham.org.
This paper is available on line at http://www.jbc.org

carcinoma cell lines that exhibited different ABO phenotypes(4). Possible allele-specific mutations were identified. Fouramino acid substitutions were discovered between A and B
transferases. O alleles were more homologous to A alleles thanto B alleles. A single base deletion was found near the N
terminus of the coding sequence in most of the O alleles, whichcaused the codon frame to shift. This resulted in a truncatedprotein without glycosyltransferase activity. In addition to thethree major alleles (A1, B, and O), we also identified mutations
that modified the enzymatic activity by determination of thepartial nucleotide sequences of subgroup alleles (A2, A3, Ax,
and B3) (5–7). We also elucidated the molecular mechanisms oftwo phenomena named cis-AB and B(A) (7, 8). Although theincidence was low, another type of O allele was discovered thatlacked the single base deletion but contained an amino acidsubstitution at the residue crucial for nucleotide-sugar recognition/binding (9). Although no functional analyses have been
performed to disprove polymorphism, others have reported additional alterations (10 –15). The nucleotide and deduced aminoacid sequences of a variety of ABO alleles are posted on the
Blood Group Antigen Gene Mutation Database developed by
Blumenfeld and colleagues (available on the World Wide Web).
A/B antigens are not restricted to humans but are widely
present in nature (16). We therefore investigated the presence/
absence of homologous sequence(s) in the genomes of otherspecies of organisms (17). Hybridization of zoo blots, using theradiolabeled human A transferase cDNA probe, showed weaksignals in chicken genomic DNA but strong signals, comparablewith the signal detected in human DNA, in genomic DNA frommice and other mammals. No signals were detected in genomic
DNA from lower species of organisms in the evolutionary tree.
We next determined the partial nucleotide sequences of the
primate ABO genes (17). The glycosyltransferases responsible
for A or B phenotypes in primates were shown to conserveamino acid substitutions corresponding to codons 266 and 268
in humans. A similar study was also reported by others (18).
Through comparative sequence analyses of the ABO genes
from humans and apes, we and others proposed a convergent
hypothesis of evolution that ABO genes arose from independent mutations after the speciation of humans and apes (19, 20).
No apparent disadvantages are recognized among any of thephenotypes involving the ABO polymorphism. Hemolytic disease of newborns may be a natural selection against specificcombinations of blood groups between the mother and fetus.
However, serious incompatibility cases are rare with ABO,
since the natural antibodies against A and B antigens aremostly IgM and do not cross the placenta. Although someanti-A, B antibodies are IgG and capable of crossing the pla-

13701

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007

We have cloned murine genomic and complementary
DNA that is equivalent to the human ABO gene. The
murine gene consists of at least six coding exons andspans at least 11 kilobase pairs. Exon-intron boundaries
are similar to those of the human gene. Unlike human A
and B genes that encode two distinct glycosyltransferases with different donor nucleotide-sugar specificities, the murine gene is a cis-AB gene that encodes an
enzyme with both A and B transferase activities, andthis cis-AB gene prevails in the mouse population. Cloning of the murine AB gene may be helpful in establishinga mouse model system to assess the functionality of the
ABO genes in the future.

13702

Murine ABO Gene Equivalent

EXPERIMENTAL PROCEDURES

Materials
Mouse genomic DNA library (ML1044j), which was constructed byreplacing the internal BamHI–BamHI stuffer fragment of the ␭ EMBL3
SP6/T7 vector with MboI-partially cleaved genomic DNA fragments of
the BALB/c strain of mouse, was purchased from CLONTECH (Palo
Alto, CA). A Marathon cDNA Amplification Kit and an AdvanTage
PCR1 cloning kit were also from CLONTECH. A GeneClean kit waspurchased from Bio101 (La Jolla, CA), and pT7T3␣18 plasmid vector,
␣-S-dGTP, ␣-S-dCTP, and S-300 MicroSpin columns were from Amersham Pharmacia Biotech. LipofectAMINE was purchased from Life
Technologies, Inc. [14C]UDP-GalNAc, [14C]UDP-galactose, and[␣-32P]dCTP were from PerkinElmer Life Sciences, and 2⬘-fucosyllactose was from Oxford Glycosystems (Rosedale, NY) and from Calbiochem. Murine anti-A and anti-B monoclonal antibody mixtures werepurchased from Ortho Diagnostic Systems (Raritan, NJ). Biotinylated
Ulex europaeus agglutinin I, Vectastain Elite ABC kit, and 4-chloro-1naphthol substrate kit were from Vector Laboratories (Burlingame,CA), and EZ-Link Sulfo-NHS-LC-Biotin was purchased from Pierce.
Bluescript SKM13⫹ vector, Prime-It II kit, Duralose-UV membranes,
and frozen competent XL1-blue strain of Escherichia coli bacteria werepurchased from Stratagene (La Jolla, CA). The ULTRAhyb hybridization buffer was obtained from Ambion (Austin, TX). Restriction endonucleases and nucleic acid-modifying enzymes were from Life Technologies, New England Biolabs (Beverly, MA), or Roche Molecular
Biochemicals. dRhodamine dye terminator cycle sequencing ready reaction kits and BigDye cycle sequencing kits were purchased from
PerkinElmer Life Sciences.

Methods
Isolation of Genomic DNA Clones and Construction of Restriction
Endonuclease Cleavage Maps—Under low stringency conditions, ⬃1
million plaques from the mouse genomic DNA library were screenedusing a human A transferase cDNA probe by the plaque hybridization
method (25). Radiolabeled probe was prepared by the random hexamer
primer method, using a Prime-It II kit and [␣-32P]dCTP (26). After four
rounds of screening, individual clones were isolated. Phage DNA wasprepared, cleaved with restriction endonucleases, gel-electrophoresed,
1
The abbreviations used are: PCR, polymerase chain reaction; ␣-SdGTP, 2⬘-deoxyguanosine-5⬘-O-(1-thio-triphosphate); ␣-S-dCTP, 2⬘-deoxycytidine-5⬘-O-(1-thio-triphosphate); RACE, rapid amplification of
cDNA ends.

and Southern transferred. Hybridization was then performed to construct restriction enzyme cleavage maps.
Subcloning, Nested Deletion, and DNA Sequencing—DNA from
MABO␾16 phage clone was cleaved with HindIII and SalI and subcloned into the Bluescript SKM13⫹ vector. Nested deletion constructs
were prepared by the ExoIII-mung bean nuclease method (27). Whereno unique 3⬘-overhang restriction sites were available, the thioderivative fill-in reactions were performed with Klenow enzyme using ␣-SdGTP and ␣-S-dCTP before ExoIII treatment. After transformation of
E. coli XL1-blue strain, plasmid DNA was prepared from individualclones and analyzed for insert size. The nucleotide sequences were
determined by Sanger’s dideoxy chain termination method using thedRhodamine dye terminator cycle sequencing ready reaction kit (28).
Sequences were aligned using Lasergene SeqMan II sequencing project
management software.
5⬘-RACE cDNA Cloning—RNA from the CMT-93 rectal carcinoma
cell line (ATCC 223-CCL), established from a C57BL strain of mouse,
was prepared and used for the 5⬘-RACE experiments (29). We followed
the Marathon cDNA amplification protocol provided by the manufacturer. Briefly, the first strand of cDNA was synthesized from RNA using
Moloney murine leukemia virus reverse transcriptase and Marathon
cDNA synthesis primer. After the second strand was synthesized with
RNase H, E. coli DNA polymerase I, and E. coli DNA ligase, theMarathon cDNA adaptor was ligated. Nested PCR was performed, firstwith AP1 adaptor primer and MY-1 primer and then with AP2 andMY-2 primers. AP1 and AP2 primers were provided in the kit. The
nucleotide sequences of MY-1 and MY-2 primers were complementary
to the sequences in the coding region of the murine ABO genes. Theirsequences were as follows: 5⬘-TTAGTTTCTGATTGCCTGATGGTCCTTGGGCAC and 5⬘-TCATGCCACACAGGCTCAATGCCGT for MY-1
and 2, respectively. PCR products were electrophoresed through a 3%
agarose gel, and the DNA was gel-purified using the GeneClean kit.
DNA fragments were then ligated with pT-Adv vector from the AdvanTage PCR cloning kit by the T-A cloning method. Nucleotide sequencesof the inserts were determined.
Preparation of Eukaryotic Expression Constructs—The BamHI–XhoIfragment containing the last coding exon of the murine ABO gene was
first subcloned from a murine ABO genomic clone, MABO␾11, into the
pT7T3␣18 plasmid vector. The BamHI site was located in the intron
preceding the last coding exon of the murine ABO gene. The XhoI site
was in the ␭ EMBL3 SP6/T7 vector next to the BamHI site used toaccommodate genomic DNA. This construct was then digested with SstIand SnaBI. The SstI site was within the pT7T3␣18 plasmid vector, andthe SnaBI site was located downstream of the stop codon of the mouse
ABO gene coding sequence. The SstI–SnaBI fragment containing thecoding sequence in the last coding exon of the mouse ABO gene wasthen isolated. The human B transferase expression construct withintron, pBBBB (30), was cleaved with BamHI, blunt-ended by the
Klenow filling-in reaction, and then digested with SstI. The SstI site
was in the intron preceding coding exon 7 of the human ABO gene;
BamHI was in the eukaryotic expression vector (originally pSG-5). The
SstI-blunt (BamHI) vector fragment containing the human B transferase cDNA sequence of exons 1– 6 was then ligated to the mouse SstI–SnaBI fragment to produce the human-mouse chimeric gene (pHumanmouse chimera). A murine cDNA eukaryotic expression construct wasthen constructed by replacing the EcoRI–AflII fragment from the chimeric construct with the EcoRI–AflII fragment from a 5⬘-RACE clone in
the pT-Adv vector. The EcoRI site of the clone was in the plasmid andlocated 5⬘ upstream of the cDNA end. The AflII was in the last codingexon. The EcoRI site in the pSG-5 vector was located downstream of the
SV40 early promoter and upstream of the human cDNA sequence. The
resultant construct (pMouse) contained the entire coding sequence ofthe mouse ABO gene cDNA.
DNA Transfection and Enzymatic Assays—Plasmid DNA was prepared by the SDS-alkaline method (25). The HeLa cell line derived froma human adenocarcinoma of uterus was used as a recipient of transient
DNA transfection analyses. The HeLa cells express H antigens on theircell surfaces and have been successfully used in similar transfection
experiments of A and B transferase expression constructs (5, 30, 31).
Following the manufacturer’s protocol, we used LipofectAMINE fortransfection. Seventy-two hours after transfection, the cells were
washed and harvested. Cell pellets were then lysed in buffer (0.1 M
NaCl, 25 mM sodium cacodylate, 10 mM MnCl2, and 0.1% Triton X-100).
A/B transferase activity was determined by measuring the transfer ofcarbon-14 from [14C]UDP-GalNAc or [14C]UDP-galactose to the acceptor substrate 2⬘-fucosyllactose, as described previously (31). After incubation, the reaction products were separated from unincorporated nucleotide-sugars by AG1-X8 anion exchange column chromatography.

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007

centa, A/B antigens are not well developed in fetuses. Therefore, little damage is done. There should be some reason for the
existence of ABO polymorphism in the population. It has been
speculated that the possible role of the ABO system is to provide resistance against infection (21). Actually, Leb (Fuc␣132
Gal ␤133 (Fuc ␣134) GlcNAc-), an ABO-related structure,
was demonstrated to be the receptor for a Gram-negative bacillus, Helicobacter pylori, a causative agent for gastritis, peptic
ulcer, and possibly gastric cancer (22). A and B transferases
modify the Leb structure into ALeb and BLeb structures, which
H. pylori does not bind to in vitro. This may explain the earlier
observation that group O individuals have a higher incidence of
stomach ulcer than individuals in any other group (21). Antibodies against the ␣133 Gal epitope (Gal ␣133 Gal-) were
demonstrated experimentally to block the interspecies infection of certain retroviruses (23). From this result, anti-A andanti-B antibodies have been suspected to play a role in inhibiting the epidemics of certain infections. Some type of selection
based on the advantage/disadvantage of having antigens/antibodies may have been operating at the ABO locus to secure thesurvival of species from extinction during evolution. The reportthat an anti-A monoclonal antibody neutralized human immunodeficiency virus particles produced by lymphocytes fromgroup A individuals but not from group B or O individuals (24)may support this hypothesis. To experimentally assess thefunctionality of the ABO genes, establishing an animal model iscritical. As an initial step, we cloned and characterized themurine ABO gene equivalent.

Murine ABO Gene Equivalent

RESULTS

Mouse Genome Contains the Human ABO Gene Equivalent—
Certain mammalian cells exhibit ␣133 Gal epitopes. The
cDNA encoding ␣1,3-galactosyltransferase that synthesizes
this epitope was cloned from cow (32), mouse (33), and pig (34,35). Humans do not exhibit this epitope but possess the antibody against the epitope in sera (36). Human sequence corresponding to this gene was shown to be a pseudogene due toframeshifts and nonsense mutations (32, 37). A/B transferases
utilize the galactose substrate with fucose, whereas ␣1,3-galactosyltransferase utilizes the substrate without fucose. ABO
genes and ␣1,3-galactosyltransferase genes share significant
homology at both the nucleotide and deduced amino acid sequence levels (30). Cloned canine cDNA encoding Forssman
glycolipid synthetase (UDP-GalNAc:globoside ␣1,3-N-acetyl-Dgalactosaminyltransferase) also exhibited sequence homology(38). Therefore, these genes are believed to have derived fromthe same ancestral gene and constitute the ABO gene family.
Southern hybridization experiments of murine genomic DNA
showed different banding patterns when murine ␣1,3-galactosyltransferase cDNA probe and human A transferase cDNA
probe were used (17, 33). Accordingly, the ABO gene equivalent
was assumed to exist in the mouse genome (17). Results fromour cloning experiments of the murine ABO gene describedhere concluded that mice actually do possess an ABO gene
equivalent.
Organization of the Murine Gene Is Similar to the Human

Counterpart—We cloned the genomic DNA sequence encompassing most of the murine ABO structural gene. By screening
1 million phage plaques from a murine genomic DNA library,
we obtained a total of nine independent clones that hybridized
with the human A transferase cDNA probe. A preliminary
mapping showed that two phage clones named MABO␾11 andMABO␾16 contained the entire coding sequence in the lastcoding exon. Since the MABO␾16 clone contained the sequence
farther upstream, this clone was used for the nucleotide sequence determination. The MABO␾11 clone containing thefarther downstream sequence was used to construct a humanmouse ABO gene chimeric expression construct as well as amurine gene expression construct. We sequenced the entire
insert in the MABO␾16 clone (⬃11.2 kilobase pairs) with morethan 99.9% accuracy. Almost all of the coding sequence wascontained in the sequenced region. The exon-intron boundaries
were determined and are shown in Fig. 1. Fig. 1, A and B,
represents two probable splicing patterns, although other possibilities still exist because the sequence encoding the first few
amino acid residues has not yet been identified. There are six
coding exons in Fig. 1A and seven in Fig. 1B. Approximately 4.0
kilobase pairs and 70 base pairs upstream of the splicing acceptor site of coding exon 2 (cEXON 2) in Fig. 1A, there was a
CTCAGAG sequence and a TGAATCTCAG sequence, respectively. These sequences may be portions of the coding sequence,since they are found upstream in the cDNA preceding thesequence in cEXON 2. Fig. 1B depicts the case where GAATCTCAG of the latter TGAATCTCAG sequence represents the sequence in the preceding exon. In that case, the acceptor site ofcEXON 3 needs to shift 2 nucleotides upstream, which would
break up the GT-AG rule of splice junctions. We determined theentire nucleotide sequence contained in the MABO␾16 clone,which included ⬃5.0 kilobase pairs of sequence upstream of the
splicing acceptor site of cEXON2 in Fig. 1A. No sequence corresponding to the 5⬘-untranslated region was found. Therefore,
the promoter region of the murine ABO genes must residefarther upstream. The sequence corresponding to human coding exons 3 and 4 was found in one exon in the mouse gene.
However, the number of amino acid residues (19 amino acids)
was much smaller than that of human exons 3 and 4 combined(35 amino acids). Further studies are needed, since there may
be an alternative splicing that would divide this small exon into
two smaller exons with an intron in between. The entire insert
sequence in the MABO␾16 clone and the entire cDNA sequence
have been deposited in the DNA Data Bank of Japan (DDBJ)(accession numbers AB041038 and AB041039).
The Nucleotide and Deduced Amino Acid Sequences of the
Mouse ABO Gene Equivalent—The nucleotide and deduced
amino acid sequences in the coding region of the murine cDNA
were aligned with those of human A1-1 (A101) allele (accession
number AF134412 in GenBankTM) by combining the Clustal
method (40) and the J. Hein method (41) using the MegAlignsoftware. Results are shown in Fig. 1. Especially high homology
was observed in the coding sequence in the last two codingexons. The percentages of identical nucleotide and amino acidresidues in the last two coding exons were 78% (642/822) and
81% (222/273) between the two species, respectively. The
amino acid sequence of the murine gene was also aligned withthe amino acid sequences of human A and B transferases,mouse ␣1,3-galactosyltransferase, and canine Forssman glycolipid synthetase. Results are shown in Fig. 2A. The percentagesof the identical amino acid residues of the coding sequences in
the last two coding exons are 47% (127 of 272) between themouse ABO and ␣1,3-galactosyltransferase genes and 49%(132 of 272) between the mouse ABO gene and the dog Forssman synthetase gene. Fig. 2B highlights the amino acid se-

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007

The incorporation of radioactivity was determined using a scintillation
counter.
PCR Amplification and Nucleotide Sequence Determination of the
ABO Gene Fragments from Several Strains of Mouse Species and Subspecies—Murine submaxillary glands were used for the expression
analyses of A/B transferases. A and B transferase activities were measured by the incorporation of carbon-14 from [14C]UDP-GalNAc or[14C]UDP-galactose to 2⬘-fucosyllactose. Although the same reaction
conditions were used, the reaction product was separated from theprecursor substrate by paper chromatography rather than column chromatography. Genomic DNA was prepared by the proteinase K-SDS
method and used to amplify a DNA fragment derived from the murine
ABO gene. The names and nucleotide sequences of the primers usedwere as follows: SN-16, 5⬘-GAGACTGCAGAACAACACTT; SN-17, 5⬘CAATGCCGTTGGCCTTGTC. The PCR-amplified DNA fragments
were purified through chromatography using S-300 MicroSpin columns
and subjected to direct DNA sequencing reactions with the BigDye cycle
sequencing kit. After the sequencing reaction, DNA was purified andthen analyzed using an ABI Prism 377 automatic DNA sequencer.
Detection of ABH Antigen Expression—Expression of the ABH antigens
in murine submaxillary glands was examined immunologically using extracts spotted on a nitrocellulose membrane. Murine anti-A and anti-B
monoclonal antibody mixtures were biotinylated using EZ-Link Sulfo-NHSLC-Biotin, following the protocol provided by the manufacturer. After biotinylation, the unincorporated biotin was removed using Microcon 30 centrifugal filter devices. The submaxillary glands from C57BL and ICR strains ofmice were homogenized in buffer containing 20 mM Tris-HCl (pH 7.5), 0.15
M NaCl, and 1% Triton X-100. After centrifugation, the supernatant was
diluted with buffer containing 25 mM Tris-HCl (pH 7.5) and 0.1% SDS. The
extract was then spotted onto a Duralose-UV membrane. As controls, theextracts similarly obtained from human colon adenocarcinoma SW48 cells(AB phenotype) and from group A and O porcine submaxillary glands werealso spotted on the membrane. After drying for 15 min, the membrane wastreated with 0.3% hydrogen peroxide and 0.3% fetal calf serum in phosphate-buffered saline for 5 min to block endogenous peroxidase activity.
After washing, the membrane was incubated overnight in phosphate-buffered saline containing 4% bovine serum albumin at 4 °C. The membrane
was then cut into four pieces, which were individually incubated with either
biotinylated murine anti-A monoclonal antibody mixture, biotinylated murine anti-B monoclonal antibody mixture, biotinylated Ulex europaeus agglutinin I, or bovine serum albumin (negative control) for 1 h at room
temperature. The filters were washed separately and then incubated collectively with the Elite ABC reagents for 15 min. After washing with phosphate-buffered saline, the membranes were treated with 4-chloro-1-naphthol substrate for color development.

13703

13704

Murine ABO Gene Equivalent

quences of the region important for the recognition/binding of
nucleotide-sugars. The phylogenetic tree is shown in Fig. 2C.
The cloned mouse gene was evolutionarily mapped closest to
the human ABO gene. It was also mapped closer to the canine
Forssman gene than the murine ␣1,3-galactosyltransferase
gene.
Murine Gene Encodes an Enzyme with both A and B Transferase Activities—We examined whether the isolated mouse
ABO gene sequence could encode a functional glycosyltrans-

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007

FIG. 1. Comparison of the exon-intron boundaries and the nucleotide
and deduced amino acid sequences of
the coding region of the murine AB
gene with those of the human A1-1
(A101) allele. The nucleotide sequence ofthe mouse gene in the MABO␾16 phageclone and the sequence of the 5⬘-RACEamplified cDNA were used to determine
the exon-intron boundaries. Almost all of
the coding sequence was contained in thecloned genomic DNA fragment. Since thesequence encoding the first few aminoacid residues is still missing, the earlier
exon-intron boundaries have not yet been
completely determined. In Fig. 1, A and B,
two possible splicing patterns are presented. There are six coding exons in A
and seven in B. In A, ⬃70 base pairs upstream of the splicing acceptor site in coding exon 2 (cEXON 2), there was a
GAATCTCAG sequence. If the acceptor
site is shifted upstream by 2 nucleotides,that sequence may represent the preceding exon sequence as shown in B. This
shift, however, breaks up the GT-AG ruleof splice junctions. In addition, no 5⬘-untranslated region sequence is found upstream. Therefore, A may represent whatmost likely occurs. XXXXXX, the nucleotide sequence is unknown. Unlike the human sequence, the sequence in humanexons 3 and 4 was contained in a single
exon in the mouse gene. The numbers
shown in parentheses next to the nucleotide numbers of the coding sequence indicate the nucleotide residue numbers of
the MABO␾16 phage clone insert deposited to DDBJ (accession number
AB041038). The nucleotide sequence ofthe murine AB gene cDNA has also been
deposited (accession number AB041039).
Also shown is the comparison of the nucleotide and deduced amino acid sequences in the coding region of the murine gene with the human A1-1 (A101)
gene sequences (accession number
AF134412 in GenBankTM). A combined
method incorporating the Clustal method(40) and the J. Hein method (41) was usedwith the MegAlign program. High homology was observed in the coding sequencein the last two coding exons. The percentages of the identical nucleotide and aminoacid residues were 78 and 81% between
the two sequences, respectively.

ferase. We first constructed a human-mouse chimeric constructin an eukaryotic expression vector pSG-5. A DNA fragment
containing coding sequence in the last coding exon of the mouse
genomic sequence was linked downstream of the human cDNA
sequence of exons 1– 6 in the human B transferase expression
construct, pBBBB (30). When DNA from the chimera construct
was transiently transfected into HeLa cells, the appearance ofboth A and B transferase activity was observed (Table I). Because there may be inactivating mutations in the upstream

Murine ABO Gene Equivalent

13705

sequence, as observed in human O alleles, we constructed amouse cDNA expression construct. The upstream sequence of
the AflII site in the last coding exon of the mouse gene in the
chimeric construct was replaced by the cDNA sequence obtained from the 5⬘-RACE experiment. This construction produced a mouse cDNA expression construct containing the entire coding sequence. Results from the enzymatic analysis of
the transfected HeLa cells are also shown in Table I. Both A
and B transferase activities were detected in the cell extract.
Based on these results, we concluded that the murine gene is
an AB gene that encodes a protein capable of utilizing both
UDP-GalNAc and UDP-galactose donor substrates to synthesize A and B antigens.
AB Gene Is Prevailing in Mice—We have determined thepartial nucleotide sequences of the coding region in the lastcoding exon of the murine ABO gene using genomic DNA from

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007

FIG. 2. Comparisons of the aminoacid sequences of the coding regions
among murine AB gene, human A1-1
(A101) and B (B101) genes, murine
␣1,3-galactosyltransferase gene, andcanine Forssman synthetase gene
and an evolutionary tree of the ABO
and related genes. A, the amino acidsequence of the mouse AB transferase
was compared with those of the human A
and B transferases, mouse ␣1,3-galactosyltransferase (mouse GalT), and canine
Forssman glycolipid synthetase (dogForssman). The mouse AB transferase
was 47 and 49% identical to the mouse␣1,3-galactosyltransferase gene and thedog Forssman synthetase gene in the lasttwo coding exons, respectively. B, the coding sequences around the amino acid residues important for the nucleotide-sugarrecognition/binding are compared. The sequences from the human O03 allele that
contains a missense mutation around thisarea (9) and the cis-AB01 allele with dualtransferase activity (8) are also included.
C, a phylogenetic tree of the ABO genefamily is shown. The following sequencesin GenBankTM were used for the computation of evolutionary distance (46) andneighbor-joining tree (39): marmoset
GalT (S71333), bovine GalT (J04989), pig
GalT (L36152), mouse GalT (M85153),
and dog Forssman synthetase (U66140).
Numbers on interior branches denotebootstrap probabilities in percent. Branch
lengths are proportional to the numbersof amino acid substitutions, and a scale isgiven at the bottom of the tree. The dottedline denotes the probable root node of thistree. This node also corresponds to a gene
duplication event producing both the␣1,3-galactosyltransferase
geneandABO-related gene. The latter lineage further experienced another gene duplication, resulting in creation of both Forssman glycolipid synthetase gene and ABO
gene. The murine AB gene was mappedclosest to the human ABO genes. It also
mapped closer to the canine Forssman
gene than the murine ␣1,3-galactosyltransferase gene.

several species and subspecies of mice. The results are summarized in Table II. Mouse strains analyzed were Mus musculus
domesticus (B10 and png2 strains), M. musculus molossinus(MSF/Msf strain), M. musculus musculus (BLG/Msf strain), M.
musculus breviostris (BFM/2Msf and NJL/Msf strains), M.
musculus castaneus (CAST/Ei and HMI/Msf strains), M. musculus subspecies (SWN/Msf strain), and M. spicilegus (ZBN
strain). Except for M. spicilegus, all others were subspecies ofM. musculus. The results identified several nucleotide substitutions, some of which resulted in amino acid substitutions.
However, none were found at the four positions that would
distinguish between the human A and B transferases. Furthermore, both A and B transferase activities were also detected in
the submaxillary gland extracts from those strains of mice thatexhibited amino acid substitutions. Therefore, there are no
mutations that change the donor nucleotide-sugar specificity in

13706

Murine ABO Gene Equivalent

the sequenced and unsequenced regions of the gene in at least
those five strains of mice examined.
A and B Antigens Are Expressed in Mice—We have shownthat the mouse equivalent of the human ABO gene encodes aprotein capable of transferring both GalNAc and galactose
through in vitro enzymatic assays using extracts from HeLa
cells transfected with the eukaryotic expression constructs ofthe murine gene. We have also shown the presence of A and B
transferase activity in the murine submaxillary glands by useof in vitro assays of tissue extracts. However, the expression of
the protein does not necessarily prove the expression of A and
B antigens, since the enzymatic reactions require the appropriate substrates and reaction conditions. Therefore, we next
analyzed the expression of A and B antigens. Two laboratory

Construct

No DNApA(arginine)
pAAAA
pBBBB
pHuman-mouse chimera
pMouse[14C]GalNAc
incorporation[14C]galactose
incorporation

cpm

cpm

⫺9
⫺30
⫹2700
⫹10
⫹660
⫹630

⫹40
⫺110
⫹75
⫹450
⫹610
⫹600

Activity

A
B
AB
AB

DISCUSSION

We have cloned murine genomic DNA containing most of thecoding sequence that was equivalent to the human ABO gene.

TABLE II
Nucleotide and amino acid substitutions in mouse species and subspecies
The nucleotide and the deduced amino acid sequences of the murine equivalents of the human ABO gene were compared among several strainsof mouse species and subspecies. The nucleotide and the deduced amino acid sequences in the region between nucleotide 360 and 807 in Fig. 1 were
examined. The nucleotide sequence substitutions are shown in boldface type, and the resulting amino acid substitutions are shown in parentheses.
A and B transferase activity was examined using extracts from submaxillary glands. ⫹ and ND denote the presence of activity and not determined,
respectively. In these enzymatic assays, A transferase activity of 0.004 – 0.093 nmol/h/mg of protein (1.287 for the positive control human gastriccarcinoma cell line MKN 45 cell extract) and B transferase activity of 0.008 – 0.030 nmol/h/mg (0.035 for the positive control human gastriccarcinoma cell line KATOIII cell extract) were detected among different strains of mice.
Transferase
activity

Nucleotide positionName

M. musculus
domesticus
BALB/c strain
B10 strain
png2 strain
M. musculus
molossinus
MSF/Msf strain
M. musculus musculus
BLG/Msf strain
M. musculus subspecies
SWN/Msf strain
M. musculus breviostris
NJL/Msf strain
BFM/2Msf strain
M. musculus castaneus
CAST/Ei strain
HMI/Msf strain
M. spicilegus
ZBN strain

418

542

543

615

616

735

802

A

B

C
C
C

G
G
G

T
T
T

A
A
A

A
A
A

A
A
A

A
A
A

⫹
ND
ND

⫹
ND
ND

C

G

T

A

A

A

A

ND

ND

C

G

T

A

A

A

A

ND

ND

C

G

T

A

A

A

A

ND

ND

C
C

G
A
(R181H)

T
T

A
A

A
A

A
A

A
A

ND
⫹

ND
⫹

C

G

C

G

G

⫹

G

C

G

G
(M268V)G(M268V)⫹C

G(T206A)G(T206A)⫹⫹A(Q140K)

G

T

A

G(T206A)

GG(M268V)⫹⫹G

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007

TABLE I
In vitro enzymatic assays of extracts from HeLa cells transfected witheukaryotic expression constructs
DNA transfection was performed using LipofectAMINE. Human A
and B transferase expression constructs, pAAAA and pBBBB (30), wereused as positive controls; nonfunctional pA(arginine) construct (31) wasused as a negative control. A and B transferase activity was determinedby measuring the transfer of 14C from radiolabeled nucleotide-sugars to
the acceptor substrate 2⬘-fucosyllactose. The values represent thecounts in cpm after subtracting the values obtained without 2⬘-fucosyllactose from the values obtained with 2⬘-fucosyllactose in the reactions. The activity of the respective constructs was summarized and isshown in the rightmost column.

strains of M. musculus domesticus mice were analyzed. We
initially examined the agglutination of murine red blood cells
using murine monoclonal antibody mixtures against A and B
antigens under the regular agglutination conditions used for
the ABO blood group typing of human red blood cells. No
agglutination was observed (data not shown). Because we observed A and B transferase activity in the submaxillary glandextract, we next performed the inhibition study, using boiled
extracts of the murine submaxillary glands. No inhibition of
the reference human red blood cell agglutination was observed,
although the treatment of murine monoclonal antibody mixtures with the control group A porcine submaxillary glandextract resulted in some inhibition (data not shown). These
results suggested that A and B antigens are not expressed inabundance if at all. We therefore examined the expression ofthese antigens using the more sensitive immunological methodof nitrocellulose spotting. As shown in Fig. 3, both A and B
antigens were detected in the murine submaxillary glands.
Apparently, higher expression was observed with A antigens
than with B antigens. This may be attributed to the decreased
availability of UDP-galactose substrate, resulting from thecompetition between B transferase and ␣1,3-galactosyltransferase for the same donor nucleotide-sugar. The low expressionof A and B antigens may be similarly explained by the competition for the same acceptor substrate between ␣1,3-galactosyltransferase and ␣1,2-fucosyltransferase. Since ␣133-Gal
epitope produced by the ␣1,3-galactosyltransferase is abundantly expressed in murine tissues, it is likely that the ␣1,3galactosyltransferase has higher affinity for the acceptor substrates than the ␣1,2-fucosyltransferase that synthesizes H
antigens.

Murine ABO Gene Equivalent

The sequence encoding the first few amino acid residues andthe promoter region sequence farther upstream remain to becloned. In humans, ABH antigens are widely expressed on avariety of cell surface molecules in many tissues, depending on
the ABO genotypes of the individuals. These include glycoproteins and glycolipids on mucous cells, nerve cells, red cells,
epidermis, and vascular endothelium. ABH antigen expression
seems to be more highly restricted in lower mammals (42, 43).
To understand the differential expression mechanism of A/B
antigens between humans and mice, cloning of the murine genepromoter region will be necessary.
Functional analysis of the murine gene has shown that thecloned murine gene is really an AB gene and encodes an enzyme with both A and B transferase activity. In our priorstudies of the human ABO gene, we determined the molecularcauses of two interesting phenomena named cis-AB (8) andB(A) (7). Human cis-AB alleles encode a protein with strong A
and weak B transferase activity, whereas B(A) alleles encode aprotein with strong B and weak A transferase activity. A mutation was found in these alleles at each of the four amino acidsubstitutions that distinguish human A transferase (AAAA)
from B transferase (BBBB). The cis-AB alleles were represented as AAAB, whereas the B(A) alleles were represented as
BABB in those four substitution positions. By employing DNA
transfection assays of A-B transferase chimeras and in vitro
mutagenized expression constructs, we showed that theseamino acid substitutions might cause changes in specificity andactivity of the enzyme (30, 31). As shown in Fig. 2B, the aminoacid residues at codons 245 and 247 of the murine gene, whichcorresponded to human codons 266 and 268 (the third andfourth positions of amino acid substitutions between A and B
transferases), were glycine and alanine. The alanine residue isidentical to that of human B transferase, but the glycine residue is much smaller than the methionine residue of human B
transferase. Therefore, the mouse enzyme is expected to possess a larger space for donor nucleotide-sugar substrate recognition/binding. Since N-acetyl-D-galactosamine is more bulky
than galactose, the increase in space may allow for the accommodation of not only the galactose portion of UDP-galactose but
also the GalNAc portion of UDP-GalNAc, which may accountfor the dual transferase activity of the murine enzyme. Dog
Forssman glycolipid synthetase contains glycine and alanine,
the same amino acid residues as those of the murine ABO gene
equivalent, at codons 261 and 263. However, the codon 264 in
the Forssman glycolipid synthetase is valine rather than phe-nylalanine in the murine AB gene. This difference in size
(valine is smaller than phenylalanine) may possibly render dog
Forssman glycolipid synthetase able to catalyze the transfer of
only GalNAc residues and not galactose residues.
To understand the meaning and role of ABO polymorphism
during evolution, the cloned murine AB gene fragments will be
useful in the production of knockout (group O) mice at the ABO
locus. Production of group A and B mice may also be possible byknocking in the genes after modifying the specificity of the
enzymes so that only A or B transferase activity is retained.
Additional manipulation(s) may be a prerequisite to enhancethe expression of A and B antigens. These may include theknockout of the ␣1,3-galactosyltransferase gene (44) or the
introduction of the ␣1,2-fucosyltransferase gene under strong
promoter (45) to either abolish or lessen the expression of the␣1,3-galactosyltransferase. These mice with different ABO
phenotypes in the same genetic background may clarify thefunctionality of the ABO system in the future.
Acknowledgments—We thank Emily N. Wang for editing the manuscript and Dr. Sandra S. Matsumoto for critically reviewing the manuscript. We are also indebted to Dr. Toshihiko Shiroishi (National Institute of Genetics, Mishima, Japan) for supplying various strains of mice.
REFERENCES
1. Yamamoto, F. (2000) Vox Sang. 78, 91–103
2. Yamamoto, F., Marken, J., Tsuji, T., White, T., Clausen, H., and Hakomori, S.
(1990) J. Biol. Chem. 265, 1146 –1151
3. Clausen, H., White, T., Takio, K., Titani, K., Stroud, M., Holmes, E., Karkov,J., Thim, L., and Hakomori, S. (1990) J. Biol. Chem. 265, 1139 –1145
4. Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990)
Nature 345, 229 –233
5. Yamamoto, F., McNeill, P. D., and Hakomori, S. (1992) Biochem. Biophys. Res.
Commun. 187, 366 –374
6. Yamamoto, F., McNeill, P. D., Yamamoto, M., Hakomori, S., Harris, T., Judd,
W. J., and Davenport, R. D. (1993) Vox Sang. 64, 116 –119
7. Yamamoto, F., McNeill, P. D., Yamamoto, M., Hakomori, S., and Harris, T.
(1993) Vox Sang. 64, 171–174
8. Yamamoto, F., McNeill, P. D., Kominato, Y., Yamamoto, M., Hakomori, S.,
Ishimoto, S., Nishida, S., Shima, M., and Fujimura, Y. (1993) Vox Sang. 64,
120 –123
9. Yamamoto, F., McNeill, P. D., Yamamoto, M., Hakomori, S., Bromilow, I. M.,
and Duguid, J. K. (1993) Vox Sang. 64, 175–178
10. Ogasawara, K., Yabe, R., Uchikawa, M., Saitou, N., Bannai, M., Nakata, K.,
Takenaka, M., Fujisawa, K., Ishikawa, Y., Juji, T., and Tokunaga, K. (1996)
Blood 88, 2732–2737
11. Ogasawara, K., Bannai, M., Saitou, N., Yabe, R., Nakata, K., Takenaka, M.,
Fujisawa, K., Uchikawa, M., Ishikawa, Y., Juji, T., and Tokunaga, K. (1996)
Hum. Genet. 97, 777–783
12. Olsson, M. L., and Chester, M. A. (1998) Transfus. Med. 8, 231–238
13. Olsson, M. L., Thuresson, B., and Chester, M. A. (1995) Biochem. Biophys. Res.
Commun. 216, 642– 647
14. Olsson, M. L., and Chester, M. A. (1996) Vox Sang. 71, 113–117
15. Olsson, M. L., and Chester, M. A. (1996) Transfusion 36, 309 –313
16. Kabat, E. A. (1956) Blood Group Substances: Their Chemistry and Immunochemistry, Academic Press, Inc., New York17. Kominato, Y., McNeill, P. D., Yamamoto, M., Russell, M., Hakomori, S., andYamamoto, F. (1992) Biochem. Biophys. Res. Commun. 189, 154 –164
18. Martinko, J. M., Vincek, V., Klein, D., and Klein, J. (1993) Immunogenetics 37,
274 –278
19. Saitou, N., and Yamamoto, F. (1997) Mol. Biol. Evol. 14, 399 – 411
20. O’Huigin, C., Sato, A., and Klein, J. (1997) Hum. Genet. 101, 141–148
21. Pittiglio, D. H. (1986) in Genetics and Biochemistry of A, B, H, and Lewis
Antigens (Wallace, M. F., and Gibbs, F. L., eds) pp. 1–56, American Association of Blood Banks, Arlington, VA
22. Boren, T., Falk, P., Roth, K. A., Larson, G., and Normark, S. (1993) Science
262, 1892–1895
23. Takeuchi, Y., Porter, C. D., Strahan, K. M., Preece, A. F., Gustafsson, K.,
Cosset, F. L., Weiss, R. A., and Collins, M. K. (1996) Nature 379, 85– 88
24. Arendrup, M., Hansen, J. E., Clausen, H., Nielsen, C., Mathiesen, L. R., andNielsen, J. O. (1991) AIDS 5, 441– 444
25. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY
26. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6 –13
27. Chang, D. W., Tartof, K. D., and Yeung, A. T. (1993) Gene (Amst.) 127, 95–98
28. Sanger, F., Nicklen, S., and Coulson, A. R. (1992) Bio/Technology 24, 104 –108
29. Barnes, W. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2216 –2220
30. Yamamoto, F., and Hakomori, S. (1990) J. Biol. Chem. 265, 19257–19262
31. Yamamoto, F., and McNeill, P. D. (1996) J. Biol. Chem. 271, 10515–10520
32. Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., andShaper, N. L. (1989) J. Biol. Chem. 264, 14290 –14297
33. Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings,
R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227– 8231
34. Strahan, K. M., Gu, F., Andersson, L., and Gustafsson, K. (1995) Transplant.

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007

FIG. 3. Immunological detection of A and B antigens in murine
submaxillary glands. The expression of A and B antigens was analyzed by the immunostaining of the extracts spotted onto a nitrocellulose membrane. The extracts were derived from the following sources:
1) human colon adenocarcinoma SW48 cells (AB phenotype); 2) group A
porcine submaxillary gland; 3) group O porcine submaxillary gland; 4)submaxillary gland from a C57BL strain of mouse; and 5) submaxillary
gland from an ICR strain of mouse. BSA, bovine serum albumin.

13707

13708

Murine ABO Gene Equivalent

Proc. 27, 245–246
35. Strahan, K. M., Gu, F., Preece, A. F., Gustavsson, I., Andersson, L., andGustafsson, K. (1995) Immunogenetics 41, 101–105
36. Galili, U., Macher, B. A., Buehler, J., and Shohet, S. B. (1985) J. Exp. Med.
162, 573–582
37. Larsen, R. D., Rivera-Marrero, C. A., Ernst, L. K., Cummings, R. D., and Lowe,J. B. (1990) J. Biol. Chem. 265, 7055–7061
38. Haslam, D. B., and Baenziger, J. U. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,
10697–10702
39. Saitou, N., and Nei, M. (1987) Mol. Biol. Evol. 4, 406 – 425
40. Higgins, D. G., and Sharp, P. M. (1988) Gene (Amst.) 73, 237–244
41. Hein, J. (1990) Methods Enzymol. 183, 626 – 645

42. Oriol, R., Le Pendu, J., and Mollicone, R. (1986) Vox Sang. 51, 161–171
43. Oriol, R., Mollicone, R., Coullin, P., Dalix, A. M., and Candelier, J. J. (1992)
APMIS Suppl. 27, 28 –38
44. Tearle, R. G., Tange, M. J., Zannettino, Z. L., Katerelos, M., Shinkel, T. A., Van
Denderen, B. J., Lonie, A. J., Lyons, I., Nottle, M. B., Cox, T., Becker, C.,
Peura, A. M., Wigley, P. L., Crawford, R. J., Robins, A. J., Pearse, M. J., andd’Apice, A. J. (1996) Transplantation 61, 13–19
45. Costa, C., Zhao, L., Burton, W. V., Bondioli, K. R., Williams, B. L., Hoagland,
T. A., Ditullio, P. A., Ebert, K. M., and Fodor, W. L. (1999) FASEB J. 13,
1762–1773
46. Kimura, M. (1983) The Neutral Theory of Molecular Evolution, Cambridge
Press, Cambridge, United Kingdom

Downloaded from www.jbc.org at KOKURITSU IDENGAKU on June 25, 2007