Progress in the study of ABO blood group system

Legal Medicine (Legal Med) 2000 ; 2 : l-6

Progress in the study of Al30 blood group system Hisao TAKIZAWA,Yoshihiko KOMINATO, Ichiro SHIMADA Department of Legal Medicine, Faculty of Medicine,

Toyama Medical and Pharmaceutical

University, Toyama 930-

01 94, Japan

(Received FebruaT25, 2000,AcceptedFebruary26, 2000) hSTRACT Progress in the study of ABO blood group system during the last three decades was reviewed according to following 5 items. 1. Structure of H-, A- and B-active saccharides isolated from the globoside fractions from human erythrocytes. galactosaminyltransferase

2. Enzyme characterization

(A-enzyme),

zyme). 3. Immunological

of a blood group A-gene specified a-N-acetyl-

and a blood group B-gene specified

(B-en-

properties of the A- and B-enzyme. 4. The cDNA structures of human blood group

ABO genes. 5. Transcriptional

regulation of the human blood group ABO genes.

KEY WORDS: AI30 blood group system, ABH antigen, Glycosyltransferase, Transcriptional

a-galactosyltransferase

Anti-enzyme antibody, ABO genes,

regulation

which linked to the core structure of sphingolipid or

Introduction ABH active substances

peptide chain’)+.

are widely distributed

cells, tissues and secretions’). the antigenic determinants

Chemical character chain degradation

blood group specific exoglycosidases the carbohydrate

structure

of

Biochemical properties of ABH blood group glycosyltransferases

was introduced from the

results of the carbohydrate

oligosaccharides

in

by

and analysis of

of blood group active

present in human milk or derived

from acid or alkaline degradation

of mucous blood

Establishment antigen

of the chemical

brought

biosynthesis

structure

of ABH

to find enzymes to catalyze

of antigen molecules.

H-antigen

the is in-

duced by the enzyme, called H-enzyme in this article, which transfers Lfucose

from the donor; guanosine

group substances in ovarian cyst and gastric lining.

diphosphate fucose (GDP fucose) to the acceptor; p

These studies defined the ABH antigenic

galactosyl

structure

residue,

and

synthesizes

the

specific

that H antigen was determined by L-fucose a( 1-2) D-

a( l-2)

galactosyl residue, and A antigen was determined

by the enzyme, called A-enzyme in this article, which

CZ(l-3)

linkage of N-acetyl-D-galactosamine

subterminal

by

to the

fl-D-galactose of H antigen, and B-anti-

linkage of H-antigenlO). A-antigen is induced

transfers N-acetyl-D-galactosamine

from the donor;

uridine diphosphate N-acetyl-D-galactosamine

gen was done by similar binding of D-galactose to the

N-acetyl-D-galactosamine)

H antigen’)+.

nal P_galactosyl residue of H antigen molecule,

Chemical

structure

human erythrocyte

of ABH

membrane

antigens

on the

was first elucidated

from the analysis of penta- to octa saccharides

ob-

synthesizes

the specific

from the donor; (UDP D-galactose)

studies re-

vealed that ABH antigens on the erythrocytes carried on the various types of carbohydrate

were

chains,

~~(1-3) linkage

and

of A-anti-

B-enzyme in this article, which transfers Dgalactose

tained by ozonolysis of blood group-active glycosphextensive

to the acceptor; subtermi-

gen’1)12)15). B-antigen is induced by the enzyme, called

ingolipid

fractions 5)6).Further

(UDP

uridine

diphosphate

to the acceptor;

D-galactose

subterminal

galactosyl residue of H antigen molecule, thesizes the specific a (l-3)

p-

and syn-

linkage of B-antigen’5)14).

2

TAKIZAWA et al.

These blood group glycosyltransferases demonstrated

March 2000

have been

in various tissues and body fluids, such

The cDNA structures of ABO blood

as gastric mucosa, submaxillary gland, bone marrow,

group genes

human milk, ovarian cyst fluid, serum, saliva and urine’6).

Hakomori’s

The enzymes to catalyze the biosynthesis of A-antigen and B-antigen require acceptor molecule with H antigenic

structure “)I@. Therefore,

Bombay pheno-

research group succeeded

rification of A-enzyme to homogeneity

in the pu-

and then de-

termined a partial amino acid sequence

of N-termi-

nal region of the enzyme*8’. And based on the amino

type lacking ABH antigen on the red cells is derived

acid sequence,

from deficiency of the enzyme to catalyze biosynthe-

cloned and sequencedZg). Thereafter,

sis of H antigen on the red cells. It is also confirmed

of ABO blood group system, such as Al, A2, A3, Ax,

in ABH secretion

B, Bx, Cis-AB and 0, have been realized

system that non-secretors

can not

the cDNA encoding

A-enzyme was the phenotypes by small

have so much amount of ABH antigen as secretors

changes in the DNA sequence,

due to produce only small amount of H-antigen in

or deletion50)5’). Molecular genetic studies of the Al

the cells of mucous secretion.

and B genes have identified

two critical single-base

substitutions which result in amino acid substitutions

Immunological property of blood group A and B gene specified glycosyltransferases Purification

i.e. base substitution

of ABH blood group glycosyltrans-

responsible for the different donor nucleotide-sugar substrate

specificity

And a single-base

between deletion

A- and B-enzymes3*).

in the Al gene, which

ferases is susceptible of molecular analysis of the en-

shifts the reading frame of the codons and abolishes

zyme. Whitehead

the functional A-enzyme, has been identified in the

et al. w developed

the method

to

purify A-enzyme by specific adsorption on Sepharose 4B and elution with uridine diphosphate. was partially

purified

with cation

B-enzyme

exchange

chro-

We obtained

partially purified A- and B-enzyme human urine and injected the re-

study

described in above section, that blood group 0 individual was supposed to lack a genetic back ground to produce

matography using CM Sepharose CL 6Bz0). from concentrated

most common 01 alleleso). The immunological

the protein

cally reactive

molecule

to be immunologi-

to A- and B-enzyme,

are compatible

with the cDNA structure of the common 01 allele.

into rabbit. Cross-react-

Moreover,

ing antibody to inhibit activity of either A- or B-en-

DNA clones

zyme was produced in the B-enzyme preparation

group Al30 locus revealed that the coding region of

spective enzyme preparation

im-

mune rabbit serumzl). Similar cross-reacting antibody also can be produced

in the A-enzyme preparation

genomic

30 kbp of the blood

Structure of 5’flanking sequence and regulation of the ABO genes

from blood group 0 individual cannot neutralize the antibody 94).Antibody to specifically in-

encompassing

human

the ABO gene was organized into seven exonss3).

immune rabbit serum22)2s).And protein preparation cross-reacting

analysis of isolated

The ABO genes are expressed

in a cell type-spe-

hibit A-enzyme activity can be introduced by process-

cific manner,

ing antiserum with appropriate

dergo drastic changes during development, differen-

zyme preparation.

absorption

of Ben-

Antibody to specifically inhibit B-

activity also can be introduced by the reciprocal proThe cross-reacting

tiation, and maturation to these physiological have

cedure of A-enzyme preparatior?‘). antibody to A- and B-enzyme

and ABH antigens

also

been

are known to un-

of normal cells. In addition processes, profound

documented

processes such as tumorigenesis.

in

changes

pathological

To clarify the regu-

was also present in the patients of ABO-incompatible

lation of the ABO gene expression, we investigated a

organ transplantation;

human genomic DNA clone, HGl,

recipient/B plant

one in a liver transplant

(0

donor) and two in bone marrow trans-

(A recipient/O

donor

and B recipient/O

which contained

exon 1 and 4.7 kbp of the 5’-flanking sequence of the ABO genes (Fig. 1)33)34).

Progress in the study of ABO blood group system

Legal Med Vol. 2 No. 1

-4kb

-2kb

.3kb

I

I

I

I 4

I

I

I

w CpG

+I

representation

I

island

of the human ABO gene transcription.

ABO gene exon 1 and flanking region. Arrow above the sequence

I

I

Tissue-specific

Fig. 1. Schematic

+Zkb

+lkb

-1kb

I

I

3

DNA methylation

The diagram

indicates

indicate the transcription

the

start site,

and a closed box is the location of the first exon in the ABO gene. The 5’ CpG island is defined in the box, dense clustering of CpG sites is in the sequence is 11.7%,

and the motifs for transcription

represent

the position and orientation

of sequence

U14574

BLAST algorithm. sequence

A minisatellite

mechanism controlling

database

composed

to elucidate

of the National

of four tandemly

factor CBF/NF-Y

the molecular

the expression of the human

ABO genes, A 4.7 kb EcoRI/NcoI 5’-upstream

frag-

and +0.6kb in which CpG density in the circle.

The open arrows

which were assigned by homology Center

repeated

in the region located between nucleotide

start site, and a transcription

In an initial attempt

between -0.7kb Spl are indicated

of the Alu sequences,

with the Alu repeat

is represented

transcription

factor

search

for Biotechnology

using

copies of a 43 bp consensus

positions-3899

and -3618 from the

is in the circle.

transcriptional

regulation of various other genes, in-

cluding Myo-D gene%). CpG island hypermethylation is also found to be often associated of specific

with transcrip-

ment flanking the coding sequence in exon 1 of the

tional inactivation

tumour

human ABO gene was subcloned into the promoter-

genes in neoplastic cellrY. Therefore,

suppresser

we have inves-

less pGL3-basic vector at the upstream of luciferase

tigated the possible role of DNA methylation

coding sequence, and a series of nested deletion con-

ABO gene expression.

structs were prepared. were performed the respective lines

(gastric

throleukemia Results

Transient

transfection

to analyse the promoter constructs

assays

activity of

using two expressor

cell

Methylation

status of the CpG island correlated

well with the gene expression in the cell lines tested; that region was hypomethylated

in some cell lines

cell line RAT0111

and ery-

which express ABO genes, whereas hypermethylated

cell line HEL) as recipients

of DNA.

in the others which do not. Constitutive

just up-

tional activity of the ABO gene promoter was demon-

cancer

demonstrated

that the sequence

stream of the transcription for the transcriptional

start site was responsible

activity of the ABO geneP.

by transient transfection taining

ence of several GC boxes just upstream of the tran-

methylase-catalyzed

scription initiation sites. However, no sequence mo-

moter

tifs are found for tissue-specific teins. Methylation

DNA binding

pro-

of cytosine residues in the dinu-

CpG may offer one such mechanism

cause it is well known to control

be-

cell type-specific

transcrip-

strated in both expressor and nonexpressor

Inspection of the promoter region revealed the pres-

cleotide

in the

the ABO gene promoter region

sequence.

in vitro methylation

of the reporter

DNA transfection,

cell lines

of reporter constructs con-

however,

moter activity when introduced

HhuI

of the pro-

constructs

prior to

suppressed

the pro-

into the expressor

gastric cancer cell line RAT0111 cells. On the other hand, in viva demethylation

of the promoter

in the

TAKIZAWA et al.

4

non-expressor

gastric cancer cell line MKN28 cells

with DNA methyltransferase

found to reside in the region located between nu-

inhibitor 5-aza-2’deoxy-

cytidine resulted in an appearance messages as well as A antigens,

cleotide positions-3899

of A transferase

and -3618 from the transcrip

tion start site (Fig. l), and to enhance the ABO gene

enzymatic reaction

transcription

products catalyzed by A transferaseS7). Taken together,

March 2000

by binding

a transcription

factor

CBF/NF-I?.

these studies suggest that DNA

Hawkins et ~1.~~)submitted

a genomic

DNA se-

methylation of the ABO gene promoter may play an

quence involving an ABO blood group gene corre-

important role in cell type-specific expression, as well

sponding

as an altered expression

during tumorigenesis.

An

group

Al

to a sequence character

determining

at GenBank

the blood

accession

No.

intriguing scenario has been proposed wherein mul-

AC000397. When the sequence of AC000397 is com-

tiple Spl sites located at both the 5’and 3’boundaries

pared to those of U22302 and AFO14105, it is noticed

of the CpG island protect the island from the spread

that the minisatellite

of methylation from the densely methylated flanking

single repeat of a 43 bp consensus

region often containing

in AC000397

is composed of a sequence,

and

Alu repeat sequencesS6)S8)39). that the 35 bp DNA segment between nucleotide po-

Because genomic region 5’to the CpG island in the

sitions-970

ABO genes also contains two Alu repeats, this model

ACOO0397. Therefore,

may fit well with the DNA methylation

morphisms concerning

genes if cell type-specific

factors,

of the ABO

in addition

to a

and

-936

Our experimental

in U22302

is deleted

in

we tried to define DNA polyto these two loci.

result revealed that there were

ubiquitous factor Spl, are taken into consideration.

two loci of polymorphism; VNTR of a 43 bp consen-

Elucidation

sus sequence

of such factors may lead to further and

more precise resolution

of a mechanism

regulating

A minisatellite peated

and they were composing

composed

of four tandemly

Haplotype

haplotypes with the ABO

alleles as shown in Fig. 2. The single-repeated

(Fig. 1).

copies of a 43 bp consensus

and an insertion/deletion

polymorphism of the 35 bp DNA segment (ABOU2),

DNA methylation and support the functional importance of DNA methylation

(ABOUl)

sequence

5’- flanking

at the ABOUl

re-

Coding Exons (from ABoU2

link to the Al

1 to 7)

19.5 kbp

-

-

-3675 5’ 1 -Q-Al Type 7 DNA segment

-3675

!

526 C

261 G -

526 G -

++t-H+M

Type 2 DNA segment

-3675

261 G

-936

4-l-B

-936

4-1-o Type 2 DNA segment

Fig. 2. A schematic The single-repeated (ABOU2*0) and ABOU2*1 B and 41-0,

representation

of haplotypes

allele at the ABOUl

linkage to the Al allele is represented (35bp

insertion)

respectively.

261 AG

consisting

locus (ABOUl*l)

of the ABOUI,

ABOU2,

526 C

and ABO loci.

and the deletion allele at the ABOU2 locus

as haplotype

l-O-Al, and ABOU1*4

alleles linkage to the B and 0 alleles are represented

allele

and the deletion al-

lele at the ABOU2 locus (ABOU2*0)

was

sequence ABOUl

locus (ABOUl*l)

(four repeats) as haplotype 41-

allele,

and ABOU1*4

(35-bp insertion) Irshaid

(four repeats)

et aZ.@) also reported

tionship

AI30

correlation

geographical

alleles,

area/ethnic

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