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Prepublished online as a Blood First Edition Paper on December 5, 2002; DOI 10.1182/blood-2002-09-2838.
TRANSFUSION MEDICINE
From the National Institute of Advanced Industrial
Science and Technology (AIST), Glycogene Function Team and Cell
Regulation Analysis Team, Research Center for Glycoscience (RCG),
Tsukuba, Ibaraki; JGS Japan Genome Solutions, Inc, Hachioji, Tokyo;
Fujirebio Inc, Frontier Research Division, Hachioji, Tokyo; Jichi
Medical School, Division of Stem Cell Regulation, Minamikawachi,
Tochigi; Amersham Biosciences KK, Tokyo; and Gifu Prefecture Blood
Center, Japan.
The human blood group i and I antigens are determined by linear and
branched poly-N-acetyllactosamine structures, respectively. In erythrocytes, the fetal i antigen is converted to the adult I
antigen by I-branching
The blood group I and i antigens (I/i antigens)
were first detected by cold agglutinating autoantibodies, and they
display a dramatic change during human development.1-4
Fetal and neonatal red blood cells (RBCs) contain i antigen but little
I antigen, whereas I antigen expression gradually increases as the i
antigen decreases after birth. RBCs from most adults and children 18 months or older exhibit I antigen and contain little i
antigen.3,4 Thus, the I/i antigens on RBCs exhibit a
reciprocal expression profile during development. The I/i antigens are
also present on the cell surface in various tissues and body
fluids,5,6 and they are recognized as histo-blood group
antigens.7
I/i antigens are defined as carbohydrate structures on
glycoproteins8 and glycolipids.9 The i
antigen epitope is a linear poly-N-acetyllactosamine chain
that has Gal
To understand how I-branched poly-N-acetyllactosamines are
synthesized, it has been of interest to investigate the I-branching enzyme, In persons with the adult i phenotype, RBCs express i antigen but
little I antigen. The frequency of the adult i phenotype, inherited as
an autosomal recessive trait, was first reported to be 5 of
22 000.1 In the i phenotype, it is thought that the lack
of I antigen is the result of IGnT enzyme inactivation. Recently,
individuals showing the i phenotype for blood group have been reported
to have missense mutations in the conventional IGnT
gene.14 These mutations were responsible for abolishment of the conventional IGnT activity and resulted in a null phenotype of
erythrocyte I antigen, although I antigen expression was not examined
in the other tissues. In previous studies, healthy quantities of I
antigen have been detected in saliva, milk, and plasma of an individual
with the adult i phenotype of erythrocytes.15,16 These
findings suggest that there may be additional IGnT genes responsible for histo-blood group I antigen, and the elucidation of the
genes should contribute to a better understanding of the blood group
I gene locus.
In this study, we report a novel IGnT gene, designated
IGnT3, and compare its expression with that of the
conventional IGnT (IGnT1) and another novel
isoform, IGnT2. All 3 isoforms consist of 3 exons and share
the second and third exons. IGnT3 transcript expression is
demonstrated in bone marrow and is induced markedly during erythroid
cell differentiation. Only IGnT3 is detected in
reticulocytes. In addition, we describe molecular genetic data on
IGnTs of an adult i pedigree, in which missense mutations
are found in the common exons 2 and 3. It is demonstrated that the novel IGnT3 may be the most feasible candidate for the blood
group I gene and that the mutations found in exons 2 and 3 abolish all 3 I-branching enzymic activity and cause the adult i
phenotype in erythrocytes.
Cloning of novel human IGnT cDNAs
Preparation of RNA from lineage-specific differentiated
hematopoietic cells and reticulocyte-enriched fraction
The reticulocyte-enriched fraction was prepared from human peripheral blood using Optiprep (Nycomed Amersham, Oslo, Norway) according to the manual supplied. Optiprep was provided as a solution of 60% (wt/vol) iodixanol in water and had a density of 1.32 g/mL. OptiPrep stock was made at first from 10 mL OptiPrep and 0.1 mL of 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-NaOH (pH 7.4), and the density solutions were prepared by diluting the OptiPrep stock with HEPES-buffered saline. The Optiprep stock was added to whole blood to adjust its density to 1.105 g/mL, and a discontinuous gradient was made with the 4 density solutions (1.100, 1.095, 1.092, 1.077 g/mL) and sample (1.105 g/mL). After centrifugation at 800g for 30 minutes, the gradient was divided into 7 equivolume fractions. The recovered top layer of the gradient consisted of erythrocytes and enriched reticulocytes. Reticulocyte was identified with supravital staining using new methylene blue,19 and the reticulocytes were counted under a microscope. The percentage in the top layer was concentrated up to 12.1% compared with whole blood (1.0%), and no other cell types, including mononuclear cells and granulocytes, were detected. Total RNA of reticulocytes was extracted with Isogen (Nippon Gene) and was used for cDNA synthesis. Quantitative analysis of IGnT transcripts in human tissues and hematopoietic cells by real-time PCR The amount of IGnT transcripts in the various tissues and hematopoietic cells was determined by the real-time PCR method, as described in detail.20,21 Marathon Ready cDNAs of various human tissues were purchased from Clontech. The standard curves for the IGnT cDNAs and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, as an endogenous control, were generated by serial dilution of a pDONR 201 vector DNA containing the IGnT gene encoding the putative catalytic domain and a pCR2.1 (Invitrogen) DNA containing the GAPDH gene. Primer sets and probes for IGnTs were as follows: forward primer for IGnT1, 5'-acaaactaatgatccatgagaagtcttc-3'; reverse primer for IGnT1, 5'-ccattatatatgccaagggaaagtc-3'; probe for IGnT1, 5'-FAM-tgcaaggaatacttgacccagagccact-MGB-3'; forward primer for IGnT2, 5'-gctatgagtacatggttcgaagcc-3'; reverse primer for IGnT2, 5'-gccgaagtctttgtggatggt-3'; probe for IGnT2, 5'-FAM-tgaagaagaggctgggttccctttagcttaca-MGB-3'; forward primer for IGnT3, 5'-aatgccagtctttttgtggga-3'; reverse primer for IGnT3, 5'-atgtagtgattctgggtcaggtaatc-3'; and probe for IGnT3, 5'-FAM-aatatattaccatcacctttgcgaagtgtcccttg-MGB-3'. For GAPDH, we used Pre-Developed TaqMan Assay Reagents and Endogenous Human GAPDH (Applied Biosystems). Primers, probes, and cDNAs were added to the TaqMan Universal PCR Master Mix (Applied Biosystems) that contained all reagents for PCR. The PCR conditions included 1 cycle at 50°C for 2 minutes, 1 cycle at 95°C for 10 minutes, and 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. PCR products were continuously measured with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The relative amount of IGnT transcript was normalized to the amount of mRNA, total RNA, or GAPDH transcript in the same cDNA. When using GAPDH as the endogenous control, absolute transcript expression values lower than 10 amol were thought to be under the detectable level, and the data were eliminated before the normalization.Volunteer samples An adult i pedigree, 3 members with the adult i phenotype and 1 member with the common I phenotype, and 4 common I volunteers served as the study subjects. All 3 adult i individuals have congenital cataracts. Informed consent was obtained from all participants in accordance with Helsinki protocol. Total RNA and Genomic DNA were prepared from their peripheral blood cells using the RNeasy Mini Kit and the QIAamp DNA Blood Mini Kit (Qiagen). Genomic DNA sequencing was performed using PCR amplicons of each first exon and the common second and third exons for IGnT1, IGnT2, and IGnT3, respectively. PCR amplifications of the first exons were achieved by using the primer pairs of 5'-taaggtctcatataactcagtggatg-3' and 5'-gggtgagaactatatatgttccagtt-3' for IGnT1, 5'-tgtagacacaggttgcagcttagca-3' and 5'-cttttgtcctgtgaacagagcggtt-3' for IGnT2, or 5'-ctgggcttagctgagcctttgcaa-3' and 5'-ctgggcttagctgagcctttgcaa-3' for IGnT3. Common second and third exons were amplified using the primer pairs 5'-ctgaagtggagaaaccctggctta-3' and 5'-aaccctggattccacagctacctt-3', and 5'-agttgtagttagtcggagagtacct-3' and 5'-tataattacgtagccaggtcctgaa-3', respectively. Moreover, we examined the cDNA sequence of the IGnT3 transcript that was amplified from the RNA samples using the primers 5'-gctttcactctgctcagcgtggtcat-3' and 5'-tacccagccatcctgttagtgtatt-3'. Then amplified fragments of IGnT3 were subcloned into the pCRII-TOPO vector using a TOPO TA Cloning Kit (Invitrogen). These purified PCR products, obtained using the MinElute PCR Purification Kit (Qiagen), and cloned purified plasmids, obtained using the QIAprep Spin Miniprep Kit (Qiagen), were sequenced using the DYEnamic ET terminator cycle sequencing kit with MegaBACE 500 (Amersham Biosciences).Expression of IGnTs in Chinese hamster ovary cells Chinese hamster ovary (CHO) cells were cultured in -minimal
essential medium supplemented with 10% fetal bovine serum and used for
overexpression experiments on IGnTs. A full open-reading frame was cloned into the pcDNA3.1/Hygromycin(+) vector from the subcloned pCRII-IGnTs, resulting in
pcDNA3.1-IGnTs. cDNA from mutated alleles
I3i1 with A nucleotide at position 1049 and
I3i3 with A nucleotide at position 1006 were
ligated to pcDNA3.1/Hygromycin(+) vector. The plasmids were stably
transfected into CHO cells using Lipofectamine 2000 (Invitrogen), and
then the transfectants were selected using 0.6 mg/mL hygromycin.
Alterations of cell surface I/i antigen expression were examined with
human monoclonal antibodies OSK-2822 for i-antigen and
OSK-1423 for I-antigen using FACScalibur (Becton
Dickinson). OSK-28 and OSK-14 were originally designated 2A-70 and
anti-I No. 068, respectively, and were generous gifts from Dr J. Takahashi, Red Cross Blood Center (Osaka, Japan).
Identification of IGnT2 and IGnT3 We found 2 IGnT-homologous regions in the human genomic sequence database using tBLASTn in NCBI (National Center for Biotechnology Information) BLAST. One existed on BAC clone RP11-360O19 (chromosome 6 complete sequence; accession no. AL139039), and the other existed on BAC clone RP11-421M1 (chromosome 6 complete sequence; accession no. AL358777) as shown in Figure 2A. The former homologous region was located upstream of the conventional IGnT-exon 1 (IGnT1 exon 1) on RP11-360O19, and the latter homologous region was located upstream of the conventional IGnT-exon 2 and 3 (IGnT1 exons 2 and 3) on RP11-421M1. These sequences of AL139039 and AL358777 were included in an NT-numbered sequence, NT_007291 (Figure 2B). We speculated that the homologous regions are the first exons of novel IGnTs and are designated IGnT2 and IGnT3, as documented in "Materials and methods." We also speculated that the transcripts for IGnT1 (conventional IGnT), IGnT2, and IGnT3 are derived by alternative splicing, sharing exons 2 and 3. Using primer pairs, as indicated by arrowheads in Figure 2A, which were designed from information on the BAC clone sequence data (RP11-360O19 and RP11-421M1), we could successfully amplify cDNA clones of IGnT2 and IGnT3 in addition to IGnT1. Comparison of the cDNA sequences with NT_007291 revealed that our 3 IGnT cDNA sequences IGnT1,
IGnT2, and IGnT3are completely identical to the
genomic data (Figure 2C). Thus, the connection between the 2 BAC
clones, RP11-360O19 and RP11-421M1, could be thought as valid. Sequence
analyses revealed that IGnT2 and IGnT3 consisted
of 3 exons like IGnT1, and 3 isoforms shared exon 2, which
encodes 31 amino acids, and exon 3, which encodes 62 amino acids
(Figure 2C). Each exon 1 of the 3 IGnTs was tandemly arranged in the order of IGnT2, IGnT1, and
IGnT3 upstream of exon 2. These 3 IGnTs are
derived from alternative splicing of a single gene, such as mouse
IGnT A and IGnT B.24 The predicted
amino acid sequence from IGnT1 exon 1 showed 65% and 66%
homology to those of IGnT2 and IGnT3,
respectively. Homology in amino acid sequence corresponding to exon 1 between IGnT2 and IGnT3 was 65%. A
Kyte-Doolittle hydropathy analysis25 revealed that 3 IGnTs showed the type 2 transmembrane topology typical of most
glycosyltransferases. Nine cysteine residues were conserved among 3 IGnTs. IGnT2 was identified as the orthologue of mouse IGnT
B,24 but IGnT3 was a novel enzyme that has not
been reported in any species.
Expression of IGnTs in CHO cells It was reported that CHO cells do not express I-antigen or IGnT activity but do express i-antigen.11,26 After the establishment of IGnT-transfected CHO cells, we analyzed the cell surface expression of I- and i-antigens. Flow cytometric examination revealed an up-regulation of I-antigen and a concomitant down-regulation of i-antigen in IGnT2- and IGnT3-transfected CHO cells and in IGnT1-transfected cells (data not shown).Distribution of IGnT transcripts in various human tissues and cells Expression levels of IGnT transcripts in various human tissues were determined by real-time PCR and are summarized in Figure 3. IGnT2 transcript was expressed in many adult and fetal tissues ubiquitously and more abundantly than the transcript for IGnT1 or IGnT3. The expression of IGnT2 was strongest in the stomach and kidney, followed by fetus, small intestine, spleen, lung, placenta, heart, fetal lung, and fetal liver. Whole brain, cerebral cortex, fetal brain, cerebellum, and skeletal muscle expressed the IGnT2 transcript at a very low level. The expression pattern of IGnT1 was similar to that of IGnT2. However, the IGnT1 transcript was more abundant in cerebellum and fetal brain than the IGnT2 transcript. In contrast, the expression pattern of IGnT3 transcript was different from that of the IGnT1 or IGnT2 transcript. IGnT3 was strongly expressed in bone marrow, heart, stomach, and small intestine, and at insignificantly low or undetectable levels in the other adult tissues and fetal tissues examined. It was noteworthy that IGnT3 was most abundantly expressed in bone marrow. In peripheral leukocytes, in contrast, IGnT2 was highly expressed but IGnT3 was undetectable.
Expression of IGnT transcripts in lineage-specific differentiated hematopoietic cells and reticulocytes Given that erythroblasts are the major constituent in bone marrow, we examined the change in the expression level of the 3 IGnTs during hematopoietic cell differentiation (Figure 4). CD34+ progenitor cells expressed the IGnT2 transcript most abundantly, and the expression levels of IGnT1 and IGnT3 were relatively low. It was of interest that the IGnT3 transcript increased dramatically during erythroid differentiation, whereas the expression of IGnT2 was slightly down-regulated. On the other hand, during megakaryocytic differentiation, IGnT3 expression did not change at all. IGnT1 was up-regulated, and IGnT2 was down-regulated to some extent. In contrast, the transcription of all IGnTs was down-regulated during myeloid differentiation.
Up-regulation of IGnT3 transcript expression during
erythroid differentiation suggested that the biosynthesis of blood
group I determinants in terminally differentiated erythrocytes resulted from the IGnT3 remaining in human erythroid-committed cells
and reticulocytes. To test this possibility, the level of
IGnT transcript was quantitated by real-time PCR using the
reticulocyte-enriched fraction. As shown in Table
1, the IGnT3 transcript
clearly remained in reticulocytes, but we could not detect transcripts
of IGnT2 and IGnT1.
Missense mutations identified in IGnT genes of adult i phenotype Molecular genetic investigations were carried out using samples from an adult i pedigree, and the results are summarized in Figure 5. First, we determined the genomic sequence of the coding regions for IGnT1, IGnT2, and IGnT3 genes using samples from adult i and common I phenotypes. In the first exons of IGnT1 and IGnT2, no mutation or polymorphism was found. Moreover, in the first exon of IGnT3, the nucleotide substitution 816G>C was observed in members of the adult i pedigree. This substitution, however, which predicts the amino acid alteration of Glu272Asp, was detected not only in the adult i phenotype but also in 2 of 4 persons with the common I phenotype who are investigators in our laboratory and volunteered for the examination of IGnT genotype. Each was heterozygous for the 816G>C substitution. The high frequency of the allele, having only the 816G>C substitution, in the population indicates that it is a nonfunctional single nucleotide polymorphism (SNP). On the other hand, nucleotide substitutions of 1006G>A and 1049G>A were observed in the common second and third exons, respectively, of IGnT genes from the adult i phenotype. Neither was found in persons with the common I phenotype. Furthermore, we examined the cDNA sequence of the IGnT3 transcript that was subcloned into pCRII-TOPO. In the adult i pedigree, individuals 2, 5, and 6 of generation II with the adult i phenotype were found to be heterozygous with I3i1/I3i3 alleles, and a member with the common I phenotype, individual 1 in generation III, was found to be heterozygous with I3/I3i1. Nucleotide substitutions 1049G>A and 1006G>A in alleles I3i1/I3i3 predict amino acid changes of Gly350Glu and Gly336Arg, respectively. We found that the 816G>C SNP and the 1006G>A mutation coexist in the same I3i3 allele. In addition, the I3i3 allele containing the 1006G>A mutation was newly found in this study.
IGnT3 with 1049G>A and 1006G>A mutations fails to express I-antigen in CHO cells As described in the previous section, the 816G>C substitution coexisted with the 1006G>A mutation in the I3i3 allele. The frequency of the allele with the 816G>C mutation alone was high in the population. To exclude the possibility that IGnT3 cDNA containing the 816G>C mutation alone is associated with the adult i phenotype, we have established CHO cells transfected with IGnT3 cDNA containing the 816G>C SNP alone (pcDNA3.1-IGnT3 [816G>C]) or the wild-type IGnT3 (pcDNA3.1-IGnT3). Those cells were examined for I/i antigen expression by flow cytometry. Cells transfected with IGnT3 (816G>C) showed the same I/i expression profile as the wild type (data not shown), indicating that the 816G>C substitution is not the I-inactivating mutation. In addition, we have established CHO cells transfected with IGnT3 cDNA containing the 1049G>A mutation alone (pcDNA3.1-IGnT3 [1049G>A]) or IGnT3 cDNA having both mutations of 816G>C and 1006G>A (pcDNA3.1-IGnT3 [816G>C/1006G>A]). On flow cytometric analysis, cells transfected with the wild-type IGnT3 exhibited a remarkable increase of I-antigen expression and suppression of i-antigen (Figure 6L,K, respectively). Such an alteration of I/i expression was not observed in the CHO cells transfected with IGnT3 (1049G>A) and IGnT3 (816G>C/1006G>A) (Figure 6E-F,H-I, respectively). Furthermore, we have analyzed CHO cells transfected with IGnT3 cDNA having the 1006G>A mutation alone (pcDNA3.1-IGnT3 [1006G>A]). Cells exhibited the same I/i expression pattern as CHO cells transfected with IGnT3 (816G>C/1006G>A) and IGnT3 (1049G>A) (data not shown). Thus, 1049G>A in I3i1, 1154G>A in I3i2, and 1006G>A in I3i3 were determined to be responsible for the inactivation of IGnT3, whereas 816G>C in I3i3 was not responsible for it. The expression of the wild-type and mutant IGnT3 transcripts in the transfected CHO cells was examined and confirmed by real-time PCR (data not shown).
The blood group I antigen determinant is synthesized by the
IGnT enzyme after the sequential action of
Missense mutations in IGnT were reported to be responsible for inactivation of the IGnT enzyme and result in a null phenotype of erythrocyte I antigen.14 However, the mutations were examined only in the IGnT1 gene in their study. On the other hand, healthy quantities of I antigen have been detected in saliva, milk, and plasma of persons with the adult i phenotype of erythrocytes.15,16 The discrepancy between blood type i and histo-blood group I antigens might have resulted from novel IGnTs, which have not cloned yet, showing a different profile of tissue distribution from conventional IGnT1. According to our own experiments of the transcript expression and the previous experimental data on the BAC clones, it is considered that 3 IGnTs are generated by alternative splicing of a single gene, sharing exons 2 and 3, which was mapped at major blood group I locus on chromosome 6. The first exon of each gene might have been generated by gene duplication. All 3 isoforms of IGnT exhibited centrally acting IGnT activities, and their substrate specificity will be described elsewhere (A.T., unpublished data, 2002). In terms of tissue distribution, IGnT1 and IGnT2 showed similar profiles, with the transcript expression of IGnT2 greater than that of IGnT1. Brain, especially cerebellum and fetal whole brain, was the only tissue in which the expression level of IGnT1 was higher than that of IGnT2. IGnT1 may play a major role in synthesizing I antigens in the brain and may contribute to the development and function of the brain. IGnT2 transcripts, already expressed in fetal tissues, may consequently contribute to tissue development. IGnT3 transcripts were not detected in fetal liver, which is a major tissue for fetal hematopoiesis. This is consistent with the i phenotype of fetal erythrocytes. The above data should be considered carefully to analyze IGnT expression in each tissue because the cell type was not identified clearly. Mouse IGnT A (mIGnT A) and IGnT B (mIGnT B) have been reported.24 mIGnT A and mIGnT B were orthologous to human IGnT1 and IGnT2, respectively, and their tissue distribution was almost the same in various tissues in mouse. However, the mIGnT A gene is regulated by a promoter that is different from that for the mIGnT B gene, though the regulatory mechanisms of these genes have not been clarified.31 It is of interest that the tissue distribution of IGnT3 is different from that of the others. IGnT3 transcript expression was noted in the stomach, small intestine, bone marrow, and heart, but the expression level was low in the other tissues. The transcript level of IGnT2 was the highest among the 3 in leukocytes, spleen, and myeloblastoid cells derived from CD34+ cells. IGnT1 and IGnT3 transcripts were not detected or the expression level was very low in such tissues or cell lineages. This suggests that I antigen in leukocytes is mainly determined by IGnT2. On the other hand, IGnT1 and IGnT2 probably determine I antigen expression in megakaryocytes. By contrast, we found that the IGnT3 transcript level was the highest among the 3 in bone marrow cells, erythroblasts derived from CD34+ cells, and reticulocytes. These observations suggest a characteristic involvement of IGnT3 in I-antigen expression in erythrocytes. Moreover, it is suggested that the discrepancy between blood group and histo-blood group I/i antigen expression can be explained by differential gene regulation of IGnT3 and the other IGnTs and C2GnT-2.11,12,15,16 Although such gene regulation mechanism has not been examined in humans, it is noteworthy that remarkable IGnT3 gene expression is induced during erythroid differentiation of CD34+ cells. The regulatory region of human IGnT3 may be different from those of IGnT1 and IGnT2 and would be in the downstream of erythropoietin signal transduction.32 We have conducted molecular genetic studies of an adult i pedigree and
have revealed that the adult i phenotype resulted from heterozygous
mutations in exon 2 (1006G>A) and exon 3 (1049G>A) in the
IGnT3 gene. The results predict the changes Gly336Arg and Gly350Glu, respectively, in IGnT3, and the mutated cDNAs failed to
express I antigen on the CHO cell surface. Although the 1049G>A and
1154G>A mutations in exon 3 have been examined by Yu et
al,14 another missense mutation 1006G>A is a novel one
presented in the current study. The genomic organization of
IGnT gene family members reported here provides us with
better understanding of the adult i phenotype From the expression profile analysis, we conclude that the most feasible candidate for the blood group I gene is not IGnT1/2 but IGnT3. It is strongly suggested that the I-antigen phenotype in erythrocytes is determined by IGnT3, whereas I-antigen expression in other tissue fluids, including saliva, milk, and plasma, is regulated by the other IGnTs and C2GnT-2. Actually, the transcript level of IGnT2 was the highest among the 3 in the mammary gland. IGnT2 may play a major role in synthesizing soluble I-antigens in milk. It is of interest that the water-soluble I blood group substance was detected in white i adults.15,16 In addition, it was reported that the adult i phenotype in Japanese persons is often associated with congenital cataracts.33 The i blood type of individuals in the present study is associated with cataracts, as described in "Materials and methods." However, it has been reported that the i blood type of white persons has a reduced association with congenital cataracts.34 Given that the frequency of the i phenotype is rare and that mutations in the IGnT gene locus may occur in a sporadic manner, there may be at least 2 different types of adult i mutation. One occurs in Asians, and the other occurs in white persons. Hence, further molecular genetic studies of the adult i phenotype may reveal that mutations develop in exon 1 of IGnT3, which inactivates only IGnT3, in non-Asian populations. In such persons, IGnT1, IGnT2, and their gene products should be intact, especially in milk, saliva, plasma, and corneal epithelium in which they are expressed. Investigations to examine whether the above hypothesis is true are highly awaited.
We thank Dr Junko Takahashi and Dr Hajime Yamano of the Red Cross Blood Center in Osaka, Japan, for providing anti-i OSK-28 and anti-I OSK-14 monoclonal antibodies. We thank Kumi Ishikawa, Shigemi Sugioka, Kahori Tachibana, and Risa Kawamoto for their technical assistance. Nucleotide sequences of human IGnT2 and IGnT3 reported in this paper have been placed in the DDBJ/GenBank/EBI Data Bank with accession numbers AB078432 and AB078433, respectively.
Submitted September 18, 2002; accepted November 19, 2002.
Prepublished online as Blood First Edition Paper, December 5, 2002; DOI 10.1182/blood-2002-09-2838.
Supported in part by the R&D Project of Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of a Technical Infrastructure for Japanese Industry) of New Energy and Industrial Technology Development Organization (NEDO).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hisashi Narimatsu, Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Central-2 OSL, 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8568, Japan; e-mail: h.narimatsu{at}aist.go.jp.
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© 2003 by The American Society of Hematology.
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| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-1,6-N-acetylglucosaminyltransferase involved in human
blood group I antigen expression
Top
Abstract
Introduction
-1,6-N-acetylglucosaminyltransferase (IGnT) during
development. Dysfunction of the I-branching enzyme may result in the
adult i phenotype in erythrocytes. However, the I gene
responsible for blood group I antigen has not been fully confirmed. We
report here a novel human I-branching enzyme, designated
IGnT3. The genes for IGnT1 (reported in 1993),
IGnT2 (also presented in this study), and IGnT3
consist of 3 exons and share the second and third exons. Bone marrow
cells preferentially expressed IGnT3 transcript. During
erythroid differentiation using CD34+ cells,
IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover, reticulocytes expressed the
IGnT3 transcript, but IGnT1/2 was
below detectable levels. By molecular genetic analyses of an adult i
pedigree, individuals with the adult i phenotype were revealed to have
heterozygous alleles with mutations in exon 2 (1006G>A; Gly336Arg) and
exon 3 (1049G>A; Gly350Glu), respectively, of the IGnT3
gene. Chinese hamster ovary (CHO) cells transfected with each mutated
IGnT3 cDNA failed to express I antigen. These findings
indicate that the expression of the blood group I antigen in
erythrocytes is determined by a novel IGnT3, not by
IGnT1 or IGnT2.
(Blood. 2003;101:2870-2876)
4 M 2-mercaptoethanol. The following
factors were added: stem cell factor (SCF; 50 ng/mL, Sigma) and
granulocyte macrophage-colony-stimulating factor (GM-CSF; 10 ng/mL,
Genetics Institute) for myeloid differentiation; SCF (50 ng/mL) and
erythropoietin (EPO, 3 U/mL; Chemicon International) for erythroid
differentiation; and SCF (50 ng/mL), thrombopoietin (TPO; 20 ng/mL),
and L-asparagine (2 mg/mL) for megakaryocytic differentiation. The purity and maturation stages of the differentiated cells were examined using Wright-Giemsa staining and flow cytometric surface marker analysis. Approximately 70% cells were differentiated into myeloid lineage by GM-CSF treatment, judging from the morphology and cell surface CD11b (C3bi receptor 
