Molecular genetic basis for I antigen expression

Molecular genetic basis for I antigen expressionL.-C. Yu1,2 & M. Lin3

1Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan3Transfusion Medicine Laboratory, Mackay Memorial Hospital, Taipei, Taiwan

The rapid conversion of the i to the I phenotype on postnatal red blood cells (RBCs)reflects the elaborate functions of cell surface branched-chain I antigenic structure.The adult i phenotype has been noted to be partially associated with congenital cat-aracts. These phenomena make the molecular genetics of the blood group I systemand the regulation mechanism for I antigen expression in postnatal RBCs intrigu-ing. It has been demonstrated that the human I locus expresses three IGnT forms,designated IGnTA, IGnTB, and IGnTC, which have different exon 1, but identicalexons 2 and 3, coding regions. The uncommon molecular genetics of the I locusoffers a new perspective of the formation and expression of the I antigen in differ-ent cells, and provides an insight into the questions derived from investigation ofthe adult i phenotype. The results obtained from molecular analysis of two adult igroups, with and without congenital cataracts, support the proposed molecularmechanism for the partial association of the two traits, namely that mutations thatlead to the production of mutant IGnTC only, may result in an adult i phenotypebut not congenital cataracts, whereas mutation events that occur in the commonexon 2 or exon 3 regions, which result in the elimination of the activity of all threeIGnT enzymes, would seem to lead to the development of both traits. This suggeststhat an I-gene defect may lead directly to the development of congenital cataracts.However, the result of the gene knock-out mouse model does not confirm this sug-gestion.

Analysis of the regulation for I antigen expression shows that i-to-I phenotypictransition during erythroid differentiation is regulated by the transcription factorCCAAT ⁄ enhancer binding protein a (C ⁄ EBPa), which enhances transcription of theIGnTC gene, consequently leading to the formation of the cell surface I antigen.Subsequent investigation further showed that phosphorylation of the Ser-21 resi-due on C ⁄ EBPa blocks C ⁄ EBPa activity on IGnTC gene induction, while dephos-phorylation of C ⁄ EBPa Ser-21 occurs in both the granulopoietic and erythropoieticprocesses, leading to stimulation of IGnTC gene expression and consequently I anti-gen formation. These results demonstrate that the formation of I antigen during thedevelopment of erythrocytes, and also granulocytes, is determined by a mechanismthat is known to be crucial to determining granulopoiesis, namely involvement ofthe transcription factor C ⁄ EBPa and Ser-21 dephosphorylation of this protein.

Key words: adult i phenotype, blood groups, CCAAT ⁄ enhancer binding proteinalpha, congenital cataracts, I antigen.

Histo-blood group I and i antigens

The blood group I and i antigens were first detected on

human red blood cells (RBCs). In 1956, Wiener et al. first

gave the name I to an antigen detected by a cold

Correspondence: Lung-Chih Yu, Institute of Biochemical Sciences, Collegeof Life Science, National Taiwan University, P.O. Box 23-106, Taipei 106,TaiwanE-mail: [email protected]

394

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STATE OF THE ART 4B-PL4 ª 2011 The Author(s).ISBT Science Series ª 2011 International Society of Blood Transfusion

agglutinating autoantibody called anti-I. They found that

RBCs of only a few people (five of 22,000) were non-

reactive to anti-I and this phenotype was called I-negative

or i. Following studies found that cord blood cells con-

tained a very weak I antigen, similar to samples of the i

phenotype. In 1960, Marsh and Jenkins described the first

cold agglutinating antibody, named anti-i, which behaved

in an opposite manner to anti-I: reacting strongly with

cord blood cells and RBCs with the i phenotype, but

weakly with normal adult RBCs, and thus established the i

antigen. The expressions of I and i antigens were found to

have a reciprocal relationship and to be developmentally

regulated. Adult human RBCs fully express the I antigen,

with only a few i antigen being present, while the i antigen

is predominately present on fetal and neonatal RBCs. After

birth, the quantity of I antigen gradually increases as the

level of i antigen falls, until the normal Ii status of adult

RBCs is reached after about 13�27 months of life. In this

context, the reverse conversion, I to i, has been shown to

be associated with several hematological disorders, includ-

ing thalassaemia, hypoplastic anemia, and acute leukemia.

Like ABH antigens, the Ii are also referred to as histo-blood

group antigens because they are not just detected on RBCs,

but are also found on the surface of most human cells and

on various soluble glycoproteins present in a number of

different body fluids, including milk, saliva, plasma, amni-

otic fluid, urine, and ovarian cyst fluid.

Through various studies the I and i antigenic determi-

nants have been elucidated. They are carbohydrate struc-

tures carried on glycolipids and glycoproteins and are

present on the interior structures of the complex carbohy-

drate chains bearing ABH and Lewis antigens. Based on

type 2 Galb1-4GlcNAc chains, the basic i and I structures

are characterized as straight and branched repeats of

N-acetyllactosamine (LacNAc), Galb1-4GlcNAcb1-3Galb1-

4GlcNAc-R and Galb1-4GlcNAcb1-3(Galb1-4GlcNAcb1-

6)Galb1-4GlcNAc-R, respectively. These glyco-chains

with LacNAc repeats are called poly-LacNAc chains. The

LacNAc repeats of the straight i chain are synthesized

by the sequential action of b-1,3-N-acetylglucosaminyl-

transferase (b3GlcNAcT) and b-1,4-galactosyltransferase

(b4GalT). Conversion of i antigen into an I-active

structure requires the activity of a third enzyme, the

I b-1,6-N-acetylglucosaminyltransferase (I b6GlcNAcT).

The activity of I b6GlcNAcT adds the decisive GlcNAcb1-

6 branch onto the straight poly-LacNAc chains, and,

with the subsequent actions of b3GlcNAcT and b4GalT,

the straight i chain can be converted into a complex poly-

LacNAc structure with I branchings. Thus the blood group

I gene has been assigned as encoding a b6GlcNAcT. The

conversion of i to I phenotype on RBCs is believed to

result from the appearance of an active I-branching

enzyme after birth.

Association of the adult i phenotype withcongenital cataracts

Most adult RBCs abundantly express I antigen; however, in

a small percentage of individuals, the RBCs are rich in i

antigen and contain a very low level of I. This phenotype is

called the adult i phenotype, and is believed to result from

a lack of I-branching transferase activity. Frequency of

adult i phenotype is very low, only few cases in thousands

or ten thousands. In spite of its rareness, adult i phenotype

has attracted much attention because it has been noted to

be partially associated with congenital cataracts.

Yamaguchi et al. first reported the association of the

adult i phenotype with congenital cataracts among Japa-

nese. Of 31 Japanese with the adult i phenotype, 29 had

congenital cataracts, while none of the I phenotype mem-

bers of these families had the same eye defect, indicating

there was no recombination between the Ii phenotype and

cataracts. The marked association of the two traits has also

been observed in Taiwanese. However, the association is

not as pronounced in white populations, with only a few

cases among adult i whites having been reported to suffer

from congenital cataracts.

The association of the adult i phenotype with congenital

cataracts can be explained either by a close linkage

between independent I-related and cataract-related genes

or by a pleiotropic effect of the gene responsible for the

adult i phenotype on the development of cataracts. Before

the identification of the blood group I gene and the molec-

ular basis of the adult i phenotype, it had been suggested

that the former hypothesis of a close linkage between two

independent genes was the more tenable one because of the

reduced strength of the association observed in white

populations.

Molecular genetic organization of the humanI locus

In 2001, our laboratory reported the molecular genetic

analysis of three Taiwanese pedigrees with the adult i phe-

notype. All adult i members of the three families, but not

any of the other common I members, suffer from congenital

cataracts. Mutation detection within two glycosyltransfer-

ase genes encoding enzymes with I-branch forming activity

(designated IGnT and C2GnT-M) was carried out as was

segregation analyses across the families. The results dem-

onstrated an association between molecular changes in the

IGnT gene and the adult i phenotype, suggesting that the

IGnT gene, first reported by Bierhuizen et al. in 1993, is the

blood group I gene. Three different molecular changes in

the IGnT gene, including two missense mutations and a

deletion of a long DNA segment, were identified as respon-

sible for the adult i phenotype in the three pedigrees. The

Molecular basis of I antigen expression 395

� 2011 The Author(s).ISBT Science Series � 2011 International Society of Blood Transfusion, ISBT Science Series (2011) 6, 394–398

IGnT gene has been recognized by the HUGO Gene Nomen-

clature Committee as GCNT2.

Despite the identification of the molecular bases of the

adult i phenotype, the molecular genetic basis of the associ-

ation of the adult i phenotype with congenital cataracts

remains obscure. The molecular factors revealed, however,

indicate that the association may result from a pleiotropic

effect of the same gene mutation but not from a linkage of

two independent genes, as it is unlikely that two rare

mutant I genes with different missense mutations would be

linked to a nearby gene that has, by chance, also mutated

in such a way as to cause the development of cataracts.

In 2003, both our research team and that of Inaba et al.

expanded the complexity of the human I locus and

demonstrated that three IGnT forms, which have different

exon 1 coding region, but identical exon 2 and exon 3

coding regions, are expressed from the I locus through

utilization of alternative promoters. The three IGnT tran-

scripts, designated IGnTA, IGnTB, and IGnTC, encode three

functional I b6GlcNAcT enzymes sharing 66% amino-acid

sequence identities. The three transcripts were found to

have different expression profiles across various human tis-

sues, and the three IGnT cDNAs do not have a common 5’

region, as demonstrated by 5’-rapid amplification of cDNA

ends analysis. This indicates that transcription of the three

IGnT forms may be determined by different DNA regulatory

regions and by different regulatory mechanisms.

The molecular genetics of the human I locus supports the

proposition that more than one I-branching enzyme exists.

The proposition that different I-branching enzymes may be

responsible for I-antigen synthesis in different tissues is

based on the observation of the presence of normal quanti-

ties of I antigen in the saliva, milk, and plasma of i adults.

Thus, the I locus with its complex molecular genetic archi-

tecture offers a new perspective on the formation and

expression of I antigen. When attempting to understand the

expression of I antigen in different tissues and cells and the

mechanisms for the appearance and disappearance of the I

antigen during developmental and oncogenetic processes,

then the functioning of the three individual IGnT forms

need to be considered.

Molecular genetic mechanism for the partialassociation of the adult i phenotype withcongenital cataracts

The molecular genetic organization of the I locus provides

an insight into the molecular background leading to the

partial association of the adult i phenotype with congenital

cataracts. Molecular analysis of the two adult i groups, with

and without congenital cataracts, was performed by our

research team, and showed that wild-type IGnTA and IG-

nTB, but mutant IGnTC, which was a result of mutations

occurring in exon 1C region, were present in all of the adult

i individuals without congenital cataracts, while all of the

individuals with both traits, adult i phenotype and congeni-

tal cataracts, possessed mutation events that occurred in

the common exon 2 and 3 regions of the IGnT locus, which

consequently resulted in a mutation that affected all of the

three IGnT forms. In addition, we analyzed the expression

of the three IGnT transcripts in reticulocytes and lens-epi-

thelium cells using reverse transcription-polymerase chain

reaction, and found that IGnTC was the only one of the

three IGnT transcripts expressed in reticulocytes, the red

cell precursors, whereas only the IGnTB transcript was

detected in the RNA sample prepared from human lens-epi-

thelium cells.

These results redefine the exact gene form responsible

for the expression of the blood group I antigen on RBCs as

IGnTC. Furthermore, these results allow us to propose a

molecular genetic mechanism for partial association of the

two traits, namely that a defect in IGnTC function leads to

the absence of I antigen in RBCs, whereas congenital cata-

racts occur in those i adults where all three IGnT-enzyme

functions are defective, but not in analogs where only the

IGnTC form is defective.

To date, nine different genetic alterations, including

eight missense point mutations and one deletion, have been

identified as mutant IGnT alleles among adult i individuals.

These analyzed adult i individuals belong to five different

ethnic populations worldwide, and the revealed molecular

bases in these i adults, with or without congenital cataracts,

are consistent with the proposed molecular genetic mecha-

nism for the association of the two traits, namely the muta-

tion event that occurs in the exon 1C region of the IGnTC

gene leads to the trait of adult i phenotype but not that of

congenital cataracts, whereas the mutation that occurs in

the common exon 2-3 region, and that consequently results

in elimination of the activities of all three of the IGnT

enzymes, may lead to both traits. These results highly sug-

gest that a defect in the I gene may lead directly to the

development of congenital cataracts, and thus it can be

deduced that I b6GlcNAcT activity may have a functional

role in lens transparency.

In a subsequent investigation, the molecular genetics of

the mouse I locus was found to have a high degree of

homology to that of the human I locus. However, IGnT-

deficient mice developed by a gene-knock out technique

did not show the development of congenital cataracts. Thus

the possibility that the I-gene defect is not the reason for

the development of congenital cataracts in adult i individu-

als can not be totally excluded based on this result. How-

ever, it is also possible that the discrepancy between the

phenotypes observed between I-gene deficient humans and

I-gene deficient mice may occur due to a difference in the

expression or function of one or more of the other

396 L.-C. Yu & M. Lin


glycosyltransferases in mice. One of these might have a sim-

ilar function in mouse lens-epithelium cells to that of IGnT

in human lens-epithelium cells. Notwithstanding the above

results from the molecular analysis of i adults, it is clear that

the direct evidence proving that an I-gene defect results in

the development of congenital cataracts is still lacking,

although the indirect evidence remains very strong.

Regulation of blood group I antigenexpression

The rapid appearance of I antigen on RBCs after birth indi-

cates that the I antigenic structure may play a significant

role in postnatal RBCs. It has been shown that the transition

from straight to branched poly-LacNAc structure leads to

drastic changes of antigenicity of the glyco-epitopes build-

ing on the terminals of poly-LacNAc chains. An evident

example is the blood group A and B antigens on fetal and

neonatal RBCs. Despite the presence of IgG anti-A and

anti-B antibodies in the serum of group O mothers, severe

hemolytic disease in the fetus and newborn (HDFN) due to

ABO-incompatible pregnancy is rare. Antibodies to carbo-

hydrate antigens are generally of low or only moderate

intrinsic affinity. However, binding of both combining sites

of an IgG antibody to the antigenic epitopes that are pres-

ent on a single carrier, a condition termed monogamous

bivalency, gives rise to an extraordinary energetic advan-

tage. This has been measured and it was found that the

binding of an IgG antibody with the bivalency is favored

over analogous monovalent attachment by a factor of 103

to 104. The enhancement factor for IgM antibody with an

even higher degree of multivalent attachments that

involves three or more of its combining sites is even greater

and in the order of 106. The straight and branched poly-

LacNAc structures on RBCs can be further modified into the

blood group A and B antigens. The paucity and spatial con-

figuration of the A and B antigens carried by the straight

poly-LacNAc chains restrain the monogamous bivalent

binding of IgG anti-A and anti-B antibodies, and thus the

straight poly-LacNAc structures on the fetal and neonatal

red cells lead to a very weak reactivity of the cells with

respect to anti-A and anti-B antibodies and function as a

defense mechanism against severe ABO HDFN. Since

branch formation by the poly-LacNAc chains on the sur-

faces of RBCs would compromise an ABO-incompatible

pregnancy, it has been proposed that there has been and is

a strong biological selection against the transition from the

straight-chain i to branched-chain I phenotype on fetal

RBCs. Nonetheless, formation of the I branching structure

on RBCs occurs, and is normally regulated to take place

after birth to prevent severe ABO HDFN, further suggesting

that the formation of the I branching structure should have

an important biological significance for postnatal RBCs.

In our recent study, we revealed that the i-to-I pheno-

typic transition during erythroid differentiation is regulated

by the transcription factor CCAAT ⁄ enhancer binding pro-

tein a (C ⁄ EBPa), which enhances transcription of the IGnTC

gene, consequently leading to the formation of the I anti-

gen.

C ⁄ EBPa belongs to a family of leucine-zipper transcrip-

tion factors. These proteins share related N-terminal trans-

activation domains, DNA binding regions, and C-terminal

leucine-zipper protein interaction domains. C ⁄ EBPa binds

as a homodimer or heterodimer with other C ⁄ EBP members

or other transcription factors. Further, critical functions

have been demonstrated in terms of regulating the balance

between cell proliferation and differentiation, particularly

in hepatocytes, adipocytes, and hematopoietic cells. In

hematopoietic system, it is well known that C ⁄ EBPa plays a

crucial role in the development of granulocytes and con-

trols granulopoietic differentiation and proliferation in a

stage-specific manner. Loss of C ⁄ EBPa function in myeloid

cells leads to blocked myeloid differentiation, resulting in

early blockage of granulocyte maturation. Furthermore,

C ⁄ EBPa gene mutations have frequently been observed in

patients with acute myeloid leukemia. In addition, it has

been demonstrated that C ⁄ EBPa regulates the repopulating

activity of hemetopoietic stem cells.

The phosphorylation status of C ⁄ EBPa is a critical factor

in the modulation of C ⁄ EBPa function, including its DNA-

binding and growth-inhibitory activities. It has been estab-

lished the function of C ⁄ EBPa in granulopoiesis is critically

affected by the phosphorylation status of the Ser-21 residue

within the protein, with phosphorylation of Ser-21 block-

ing the ability of C ⁄ EBPa to induce granulopoiesis. In our

study, the I antigen-activation activity of C ⁄ EBPa in ery-

throid differentiation also appeared to be modulated at a

post-translational level, and dephosphorylation of C ⁄ EBPaat Ser residues was found to be accompanied by an

enhancement of DNA binding of C ⁄ EBPa to its binding

motif in the IGnTC promoter region and by the induction of

IGnTC gene expression. Subsequent investigation showed

that phosphorylation of C ⁄ EBPa Ser-21 blocks C ⁄ EBPaactivity on IGnTC gene induction, while dephosphory-

lation of C ⁄ EBPa Ser-21 occurs in both the granulopoi-

etic and erythropoietic processes, leading to stimulation

of IGnTC gene expression and consequently I antigen

formation. These results indicate that the regulation

of I antigen formation during the erythropoietic and

granulopoietic processes share a common mechanism,

with dephosphorylation of Ser-21 on C ⁄ EBPa playing a

critical role.

With the exception of the granulocytic lineage, C ⁄ EBPahad not been implicated in the differentiation of other

hematopoietic lineages. Thus, the finding that I antigen

formation in erythroid differentiation is determined by

Molecular basis of I antigen expression 397


C ⁄ EBPa was somewhat unexpected. However, it is well

known that C ⁄ EBPa is a transcription factor with multiple

functions and multifaceted character. The variety of

C ⁄ EBPa functions is manipulated in a number of ways,

including translational control by alternative use of

initiation codons, interaction with various transcription

factors, and phosphorylation-mediated changes in DNA-

binding activity, transactivation potential, and nuclear

localization.

It will be of particular interest in future investigations to

discover whether erythroid and granulocytic lineage cells

utilize the same cellular mechanisms to regulate the phos-

phorylation status of C ⁄ EBPa. Furthermore, it is believed

that a cellular mechanism in the adult erythrocyte-

lineage cells is able to modify the status of C ⁄ EBPa

(phosphorylation or some other undetermined modifica-

tion ⁄ mechanism) such that it affects the factor’s ability to

activate the IGnTC gene, which then leads to I branching

formation. It also appears reasonable to suggest that these

cellular mechanisms may not be present in, or be active in,

fetal erythrocyte-lineage cells, and that these mechanisms

are regulated to be active only until birth. Elucidation of

these detailed cellular mechanisms will provide informa-

tion for further investigation of the molecular mechanisms

triggering the postnatal i-to-I phenotypic transition on

RBCs.

Disclosures

No potential conflict of interests to declare.

398 L.-C. Yu & M. Lin


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Molecular genetic basis for I antigen expression