From INSERM U76, Institut National de la Transfusion
Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France and
§ University of Texas-Houston Medical School,
Houston, Texas 77030
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ABSTRACT |
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The Kidd (JK) blood group is carried by an
integral membrane glycoprotein which transports urea through the red
cell membrane and is also present on endothelial cells of the vasa
recta in the kidney. The exon-intron structure of the human blood group Kidd/urea transporter gene has been determined. It is organized into 11 exons distributed over 30 kilobase pairs. The mature protein is encoded
by exons 4-11. The transcription initiation site was identified by
5'-rapid amplification of cDNA ends-polymerase chain reaction at
335 base pairs upstream of the translation start point located in exon
4. The 5'-flanking region, from nucleotide 837 to
336, contains
TATA and inverted CAAT boxes as well as GATA-1/SP1 erythroid-specific
cis-acting regulatory elements. Analysis of the
3'-untranslated region reveals that the two equally abundant erythroid
transcripts of 4.4 and 2.0 kilobase pairs arise from usage of different
alternative polyadenylation signals.
No obvious abnormality of the Kidd/urea transporter gene, including the 5'- and 3'-untranslated regions, has been detected by Southern blot analysis of the blood of two unrelated Jknull individuals (B.S. and L.P.), which lacks all Jk antigens and Jk proteins on red cells, but was genotyped as homozygous for a "silent" Jkb allele. Further analysis indicated that different splice site mutations occurred in each variant. The first mutation affected the invariant G residue of the 3'-acceptor splice site of intron 5 (variant B.S.), while the second mutation affected the invariant G residue of the 5'-donor splice site of intron 7 (variant L.P.). These mutations caused the skipping of exon 6 and 7, respectively, as seen by sequence analysis of the Jk transcripts present in reticulocytes. Expression studies in Xenopus oocytes demonstrated that the truncated proteins encoded by the spliced transcripts did not mediate a facilitated urea transport compared with the wild type Kidd/urea transporter protein and were not expressed on the oocyte's plasma membrane. These findings provide a rational explanation for the lack of Kidd/urea transporter protein and defect in urea transport of Jknull cells.
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INTRODUCTION |
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The Kidd blood group system (JK) is defined by two
codominant alleles, Jka and Jkb,
of similar frequency (0.51 and 0.49, respectively) in the Caucasian population, but showing large differences in other ethnic groups (1,
2). Alloantibodies against the Jk antigens may occasionally be involved
in severe transfusion incompatibilities and newborn hemolytic disease.
There are three common phenotypes Jk(a+b), Jk(a
b+), and Jk(a+b+)
and a rare null phenotype, Jk(a
b
), first described by Pinkerton
et al. (3), also called Jknull. The frequency of
this phenotype is increased in certain populations (Asian, Polynesian,
or Indian extraction). The Jknull phenotype results from
two different genetic backgrounds: (i) homozygous inheritance of a
"silent" allele Jk at the JK locus and (ii)
inheritance of a dominant inhibitor gene In(Jk), unlinked to
the JK locus (1, 2). Following immunization by transfusion
or pregnancy, Jknull individuals may produce an antibody
called anti-Jk3 (or anti-Jkab), which reacts
with all common red cells carrying the Jka and/or
Jkb antigens, but is unreactive with Jknull
cells themselves.
The discovery that red cells from Jknull individuals exhibited an increased resistance to lysis in aqueous 2 M urea (4) led to the suspicion that the Jk antigens might be related to the urea transporter of the human erythrocytes (for a review, see Moulds (5)). This prediction was fully confirmed by molecular cloning of the human erythroid urea transporter (clone HUT11) (6), by cross-hybridization with a rabbit cDNA transporter (7), and the demonstration that the HUT11 urea transporter and the Kidd blood group are carried by the same protein (8). This is based on the following findings: (i) in coupled transcription-translation assays, the HUT11 cDNA directed the synthesis of a 36-kDa protein, which was immunoprecipitated by a human anti-Jk3 antibody; (ii) the anti-Jk3 immunoprecipitated also a protein material of similar mass from all red cell membranes (after N-glycanase treatment), except those from Jknull cells; (iii) a rabbit antibody against the HUT11-protein reacted on immunoblots with all human erythrocytes except those from Jknull; and (iv) the structural gene encoding HUT11 was assigned to chromosome 18q12-q21 by in situ hybridization, like the Kidd blood group locus. More recently, the Kidd blood group Jka/Jkb polymorphism (D280N) was determined and used to demonstrate its lack of association with type 1 diabetes mellitus (9).
The Kidd/HUT11 polypeptide is expressed on human red cells as well as on the endothelial cells of vasa recta in the inner and outer medulla of the kidney (10, 11). Rapid urea transport may help to preserve the osmotic stability and deformability of the red cells and to stabilize osmotic gradients in the renal medulla (12, 13). In the kidney, this transport system contributes to the urinary concentrating mechanism (14, 15) involved in water preservation. Recently, a new urea transporter specific for the human kidney (clone HUT2) was cloned (16) and functionally compared with the erythroid transporter (17). These transporters are analogous to those found in other species, and their characterization, probably as a new family of transporters, will contribute to the clarification of their critical role in the renal water conservation mechanism (see Hediger et al. (18) for review) (19, 20). In this report we have further characterized the gene encoding the erythroid Kidd/HUT11 polypeptide, and we have analyzed the molecular basis of the Jknull phenotype from two unrelated individuals.
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MATERIALS AND METHODS |
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Blood Samples and Reagents-- Blood samples from individuals of common Jk phenotypes came from the Institut National de la Transfusion Sanguine (Paris, France). Two Jknull blood samples were investigated; donor L.P. was from the Center National de Référence sur les Groupes Sanguins in Paris, and donor B.S. was from the Irwin Memorial Blood Center (San Francisco, CA). Restriction endonucleases, modifying enzymes, pUC vectors, and N-glycosidase F (PNGase-F purified from Flavobacterium meningosepticum) came from New England Biolabs (UK). Radiolabeled nucleotides, [14C]urea (1.96 GBq/mmol), and [3H]raffinose (188.7 GBq/mmol) were purchased from Amersham (Bucks, UK). Expand High Fidelity and Expand Long Template PCR1 systems from Boehringer Mannheim (Germany) were used for PCR amplification. Nucleotide sequences were determined on both strands by the dideoxy chain termination method (Sanger) with Sequenase version 2.0 (U. S Biochemical Corp., Cleveland, OH) or with ThermoSequenase fluorescent labeled primer cycle sequencing kit from Amersham (Bucks, UK) using 5'(Cy5)-primers (Genset, France).
Isolation of Human JK Gene-- Approximately 2.5 × 106 phages from a human leukocyte genomic library constructed in EMBL3 Sp6/T7 (CLONTECH Laboratories, Inc., Palo Alto, CA) were plated and hybridized under standard procedures with a 32P-labeled full-length HUT11 cDNA probe (1-1528) using the random primed DNA labeling kit (Boehringer Mannheim, Germany). The positive clones were initially mapped by SacI digestion followed by Southern blot hybridization using HUT11 and the 5'-end region (see below) as probes. The positive individual gene fragments were subcloned into pUC vector and sequenced. The exons, identified from the HUT11 cDNA sequence, and their flanking regions, were fully sequenced as were some short introns. The introns were sized by PCR using primers from flanking regions of the intron. At least, all sizes were confirmed by PCR using human genomic DNA as template and Expand Long Template PCR system.
5'- and 3'-End Region Determination by cDNA Cloning--
The
5'-UT sequence was cloned by 5'-rapid amplification of the cDNA
ends (RACE) (16), using the human fetal liver 5'-RACE Ready cDNA
kit with Advantage KlenTaq Polymerase Mix
(CLONTECH). Briefly, a hemi-nested PCR
amplification under stringent conditions (94 °C for 45 s,
60 °C for 45 s, 72 °C for 1 min, for 30 cycles) was carried
out between the primer complementary to 5'-end anchor and two antisense
primers from the 5'-coding sequence of HUT11 cDNA, AS-1 (antisense
primer, position 109-86) and AS-2 (antisense primer, position 44-21).
After agarose gel analysis, transfer, and Southern blot hybridization
using 32P-labeled probe Pr.2* (nt 194 to
174),
5'-CTACCTAAAATAAAGATTATA-3', deduced from the 5'-end of an unpublished
human bone marrow cDNA clone, the positive bands were subcloned and
sequenced. Similarly, the 3'-UT sequence was amplified using human bone
marrow Marathon-Ready cDNA (CLONTECH) between
the primers complementary to 3'-end adaptor and two sense primers from
the 3'-coding sequence of HUT11 cDNA, SP-1 (sense primer, position
1219-1242) and SP-2 (sense primer, position 1304-1327. For primer
designation, nt +1 was taken as the first nucleotide of the HUT11
initiation codon (6).
Northern Blot Analysis--
Poly(A+) RNA from human
fetal liver (CLONTECH) or total RNAs isolated from
six injected oocytes according to Chomczynski and Sacchi (21), were
resolved by electrophoresis on 6% (w/v) formaldehyde, 1% (w/v)
agarose gel, and transferred to nylon filters (Zeta-probe GT, Bio-Rad).
Hybridization with 32P-labeled probes was carried at
65 °C in 0.25 M Na2HPO4, 7%
(w/v) SDS. Stringent washes were performed in 0.02 M
Na2HPO4, 1% (w/v) SDS at 65 °C for 30 min
and exposed to Biomax-MR film with intensifying screens at
80 °C.
Southern Blot Analysis-- Total genomic DNA from B lymphoid cell lines (Epstein-Barr virus-transformed) or from leukocytes was digested with SacI restriction enzyme according to the supplier (10 units/µg of DNA), resolved by electrophoresis in 0.8% (w/v) agarose gel and transferred to a nylon membrane (Hybond N+, Amersham, UK). The blot was prehybridized in 0.25 M Na2HPO4 (pH 7.2), 7% SDS for 5 min at 65 °C and then hybridized using a full-length 32P-labeled cDNA probe (exon 1-5' to end exon 11) in the same buffer. Hybridization was carried out overnight at 65 °C, and the last washing was in 0.02 M Na2HPO4 (pH 7.2), 3% SDS for 20 min at 65 °C.
Amplification by Reverse Transcription-PCR of Jk
cDNAs--
Five micrograms of total reticulocyte RNA extracted by
the acid-phenol-ammonium method (22) were used to produce the first cDNA strands using the first strand cDNA synthesis kit
(Pharmacia, Uppsala, Sweden). One sixth of the cDNA products was
used to perform a hemi-nested PCR (94 °C for 30 s, 60 °C for
30 s, 72 °C for 1 min 15 s, for 30 cycles) in a first step
between primers SP-A (sense primer, position 21 to
1) and AS-B
(antisense primer, position 1260-1237). The second PCR was performed
with 1/25 of the first PCR products in the same conditions, using
primers SP-A and AS-C (antisense primer, position 1234-1211). Final
PCR products were identified by Southern blot analysis, subcloned, and
sequenced on both strands, using an automated Alf-Express sequencer
(Pharmacia, Uppsala, Sweden).
Analysis of Splice Sites-- Direct PCR amplification was carried on genomic DNA (100 ng) under stringent conditions (94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, for 30 cycles) between primers designed from intronic sequences flanking each exon. The PCR products were subcloned and sequenced. To avoid PCR artifacts, sequencing was performed on both strands using independent PCR reactions.
Transcription-Translation and Immunoprecipitation
Assays--
Full-length (Jk+) and spliceoforms
(Jk(
6) and Jk
(
7)) cDNAs were
subcloned into the EcoRV-digested pT7TS plasmid (kindly
provided by P. Krieg, Austin, TX) and placed under the control of the
T7 promoter. The corresponding proteins were synthesized in
vitro in the transcription-translation coupled reticulocyte lysate
kit from Promega (Madison, WI) in the presence of
L-[35S]methionine (1.85 Gbq/mmol, Amersham,
Bucks, UK) and immunoprecipitated with the human anti-Jk3
antiserum obtained from an immunized Jk(a
b
) individual and with an
affinity-purified polyclonal antibody raised against the N-terminal
region (residues 8-22) of the Kidd/urea transporter polypeptide
(anti-HUT11), as described previously (8). The immunoprecipitates were
analyzed by SDS-polyacrylamide gel electrophoresis (15% separating
gel) on a discontinuous buffer system (23), followed by enlightening
treatment (NEN Life Science Products) and autoradiography.
Oocyte Urea Flux Measurements and Immunoblotting
Analysis--
After linearization of the pT7TS-cDNA constructs
with SmaI restriction enzymes, capped sense RNAs were
synthesized using T7 RNA polymerase from the mCAP mRNA capping kit
(Stratagene, La Jolla, CA). Expression studies were carried out by
microinjection of cRNAs (40 ng/oocyte) in collagenase-treated
Xenopus oocytes (24) and functional tests were realized 3 days after injection as described previously (6). To determine the
expression of Jk+, Jk(
6), and
Jk
(
7) isoforms, fractions enriched for oocyte plasma
membranes were prepared from 25 oocytes, as described by Wall and Patel (25). N-Glycosidase F treatments were performed according to the manufacturer, and a control reaction was carried out in enzyme-free buffer in otherwise identical conditions. Untreated and
N-glycosidase F-treated plasma membranes, equivalent to six
oocytes, were separated by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membranes, and incubated with the
affinity-purified antibody anti-HUT11 (2 µg/ml). Specifically bound
antibodies were detected with affinity-purified goat anti-rabbit IgG
conjugated to horseradish peroxidase (Sigma) (1:15,000 dilution) and
using Luminol/Enhancer (Pierce) according to the manufacturer's
protocole.
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RESULTS |
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Structure of the JK Gene--
Of the seven positive clones
isolated from partially Sau3A-digested human leukocyte
genomic DNA library, three Jk2,
Jk10, and
Jk12 were used to
characterize the exon organization and to define the intron/exon
junction sequences (Fig. 1).
SacI restriction mapping was established by Southern blot
hybridization with the HUT11 cDNA and the 5'- and 3'-end regions
(see below) as probes. Sequence analysis of the SacI
fragments from the three genomic clones revealed that the human
JK gene was composed of 11 exons distributed over 30 kb of
DNA (Fig. 1A). The exon sizes ranged from 50 bp (exon 10) to
551 bp (refer to the first used polyadenylation signal). The three
first exons include the 5'-UT part of the gene (see below) and the
remaining eight exons the open reading frame. The translation
initiation codon was located in exon 4 and the stop codon in exon 11. Thus, exons 4-11 corresponded to amino acids 1-50, 51-113, 114-156,
157-221, 222-270, 271-315, 316-332, and 333-389, respectively
(Fig. 1B). The intron sizes determined by PCR, ranged from
0.6 kb to approximately 8.6 kb. After partial sequence of the introns,
all exon/intron junctions were found to contain the canonical 5'-donor
gt and the 3'-acceptor ag sequences (Fig.
1B). Each class of intron-exon boundary was found in the JK gene. When all exons were sequenced and compared with the
published HUT11 cDNA isolated from a human bone marrow library, two
differences, one amino acid change (K44E) and one dipeptide deletion
were noted in exon 4 and 8, respectively. These changes correspond to
nonallelic differences previously reported between HUT11 and the
Jka/Jkb alleles (9).
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Characterization of the 5'-End of the Jk Transcript--
The
5'-end of the Jk transcript was cloned by a modified rapid
amplification of cDNA ends technique, using a fetal liver cDNA as matrix, and hemi-nested PCR between the 5'-end anchor (sens primer,
49 bp) and the AS-1 and AS-2 antisense primers (see "Materials and
Methods"). When the amplification products were analyzed by Southern
blot hybridization with the probe Pr.2* (nt 194 to
174), two bands
of ~270 and ~430 bp were detected, indicating the presence of an
alternative splicing event (Fig. 2).
These bands, which corresponded to the first ~220 and ~380 bp,
respectively, at the 5' end of the primer AS-2 (after deduction of the
49 bp from the 5'-anchor primer), were subcloned, and five independent
clones were sequenced. The results indicated that the ~430-bp
fragment included the full- length transcript and located the
transcription initiation site at 335 nt upstream from the translation
initiation codon (Fig. 2). The ~270-bp fragment, however, which was
generated by alternative splicing lacked 157 nt corresponding to exon 3 (Fig. 2). This was confirmed by Southern blot with an exon 3-specific probe which hybridized with the ~430-bp but not the ~270-bp
fragments (not shown). All of this information was used to deduce the
structure organization of the three first exons of the JK
gene described above. Further studies (reverse transcription-PCR) of
the transcripts isolated from normal human reticulocytes indicated the
presence of alternatively spliced transcripts lacking sequences encoded by exons 8 and 9, and exons 7-9 (data not shown).
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Characterization of the 3'-UT Region of the Jk
Transcripts--
The transcription start site of the JK
gene is short (335 bp upstream of the translation initiation site) and
cannot account for the size difference between the two major
transcripts (~2.0 and ~4.4 kb) detected in erythroid tissues by
Northern blot (16). Therefore, we examined whether these differences
arose from the 3' end of the Jk transcripts. At first, two unconsensus
polyadenylation signals (AATTAAA and TATAAA) and four consensus sites
(AATAAA) have been located by sequence analysis of 3'-RACE-PCR
amplifications performed with reticulocyte transcripts (see
"Materials and Methods") and by sequence analysis of the 3'-end
from the Jk12 genomic clone (Fig. 3).
Then, Northern blot analysis using the HUT11 probe as well as probes
p1, p2, and p3 located in the 3'-UT region of the JK gene
clearly indicated that the two erythroid transcripts of 4.4 and 2.0 kb
arose from differential usage of two distinct polyadenylation signals
which are separated by 2.0 kb. Indeed, the HUT11 probe located 5' to
the proximal signal AATTAAA hybridized both with the 4.4- and 2.2-kb
transcripts, while probes p1, p2, and p3, located downstream this
signal hybridize only with the 4.0-kb species (Fig. 3). The colinearity
of the full 3'-end region (exon 11) was confirmed by hemi-nested
reverse transcription-PCR and hybridization of the 2.7-kb reaction
product with the HUT11 probe (see Fig. 3).
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The JK Gene Is Not Rearranged in Jknull Cells--
In
preliminary investigations, we confirmed by Western blot analysis with
the affinity-purified anti-HUT11 antibody (nonglycosylation dependent) (10), that the Jknull cells under study
(B.S. and L.P.) lack the Jk erythrocyte membrane protein (45-69 kDa),
as seen in Fig. 4A, as well as
the Jka and Jkb antigens (data not shown).
Next, we demonstrated by Southern blot hybridization of genomic DNA an
identical SacI digestion pattern of the Jknull
samples as compared with common Jk(a+b) and Jk(a
b+) phenotypes
(Fig. 4B), indicating that a gross rearrangement of
JK gene did not occur in these variants. The five detected bands (ranging from 2 to 7 kb) are fully concordant with the known restriction sites in the JK gene (see Fig. 1) and altogether
contain all 11 exons (Fig. 4A). Moreover, restriction
analysis with a number of other enzymes showed a simple pattern
consistent with a single copy gene (data not shown). Further analysis
by DNA genotyping (9) indicated that both Jknull
individuals were homozygous for a silent Jkb allele
(data not shown).
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Molecular Analysis of the Jknull Mutations--
To
determine the molecular basis of the Jknull phenotypes,
total reticulocyte RNA from donors B.S. and L.P. were prepared and used
to amplify full-length Jk transcripts. Three amplification products of
different size (not shown) were obtained from the B.S. sample, and
several independent clones of each size were sequenced. We identified
spliceoforms that resulted from the alternative splicing of exon 6 (Jk(
6)), exons 6, 8, and 9 (Jk
(
6, 8, and 9)) and exons 6-9 (Jk
(
6-9)). The nucleotide
sequence of the 5/7 exon junction found in the Jk
(
6)
transcript is shown in Fig. 5. All of
these spliced transcripts corresponded to the spliceoforms found in
normal Jk+ individuals (see above), which in addition, all
lacked exon 6 sequences. The Jk
(
6) clone carried the
nt 838A polymorphism typical of the Jkb allele (9),
which confirmed the results from the DNA genotyping (see above). No
other alteration of the nucleotide sequence was detected. Since exon 6 was missing in all transcripts, the intron/exon junctions surrounding
this exon were analyzed on the genomic DNA prepared from donor B.S. We
found that the skipping of exon 6 was most likely caused by a G
A
transition that affected the invariant G residue of the 3'-acceptor
splice site of intron 5 (Fig. 4C). The transcripts from B.S.
potentially encode truncated polypeptides; the predicted
Jk
(
6) polypeptide would lack amino acid residues
114-156 (encoded by exon 6) of the third and fourth transmembrane
domains of the Kidd/urea transporter. The other spliceoforms
(Jk
(
6, 8, and 9) and Jk
(
6-9)) would
encode much smaller peptides with a new C-terminal extension generated
by a frameshift and premature termination.
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Expression and Functional Analysis of Jk(
6) and
Jk
(
7) Protein Isoforms--
To understand why
Jknull red cells lack Jk polypeptides and exhibit a
defective urea transport activity, we examined whether the
Jk
(
6) and Jk
(
7) transcripts
characteristic of these cells could be expressed in vitro
and in vivo.
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DISCUSSION |
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The studies reported here define the structural organization of the JK gene which encodes the human Kidd blood group/urea transporter protein. This gene spans 30 kb of DNA and consisted of 11 exons, of which exons 4-11 contained all the coding information for the mature protein. Exons 4-11 appear as being distributed along the gene into two groups of two times two exons separated by a large intronic sequence (E4, E5 and E6, E7, then E8, E9 and E10, E11), evocative of an internal gene duplication. Indeed, this may parallel the topology of the Jk polypeptide which can be subdivided into two homologous hydrophobic parts, each carrying a LP box (LPXXTXPF) encoded by exons 7 and 11, respectively, and previously reported to be an internal duplicated signature sequence of urea transporters (26).
The erythroid transcription initiation site of the JK gene was identified 335 bp upstream from the translation initiation site. Examination of nucleotide sequences that are immediately upstream revealed a typical TATA box, one inverted CAAT box at the expected position, and several putative cis-regulatory elements that may bind a variety of transcription factors (27), among which are those involved in erythroid/megakaryocytic expression (28, 29). The presence of a potential binding site for a NF-ATp factor which regulates the inducible expression of several cytokine genes is intriguing (30, 31). However, functional analysis will be required to determine which elements and which factors bind to this promoter in tissues were the Kidd/urea transporter is expressed.
We have also shown that the 4.4- and 2.0-kb erythroid Kidd/urea transport mRNAs detected by Northern blot (16) arise from usage of two different polyadenylation signals, indicating that the two equally abundant transcripts encode the same 45-kDa polypeptide. This is at the opposite to UT1 and UT2 urea transport transcripts from rat kidney (4.0 and 2.9 kb, respectively), which are alternative splice products derived from a single gene by differential utilization of alternative 5'-exon groups (19). The rat homologue of HUT11, called UT3 (20), is encoded by a single 3.8-kb transcript, which is translated into a protein of 384 amino acids sharing 80% identity with HUT11. It is possible that the first polyadenylation signal used in the human primary transcript is absent or not used in the rat. The role of large 3'-UT sequences in some transcripts is not well understood, although some may play a role in regulation of expression (32, 33).
We next examined blood from two rare unrelated Jknull
individuals, one Caucasian (L.P.) and another of Chinese (B.S.) origin, that lacked Jk antigens and Jk protein expression on red cells. Genomic
DNA analysis indicated that both donors were homozygotes for a
Jkb allele that exhibited no alteration of the
coding sequence. Therefore, although the JK genes were
present in these variants, they were not phenotypically expressed.
Sequence analysis of reticulocyte transcripts from B.S. and L.P.
indicated that alternatively spliced transcripts lacking at least exon
6 and exon 7, respectively, were present. Examination of exon/intron
junctions of the JK gene further revealed that B.S. and L.P.
were homozygous for point mutations at conserved 3'-acceptor
(ag aa) and 5' donor (gt
tt) splice sites of introns 5 and 7, respectively. Splice
site mutations lead to exon skipping and are well known to abolish or
reduce normal splicing (see Maquat (34) and references therein). Since
the Kidd/urea transporter protein is absent from Jknull cells, it is likely that the spliced transcripts are either unstable and not translated, or the corresponding truncated proteins are misrouted. At first, we found that the Jk
(
6) and
Jk
(
7) transcripts typical of Jknull cells
could be translated into polypeptides of 31 and 17 kDa, respectively,
as seen by immunoprecipitation with specific antibodies in a cell-free
transcription-translation coupled system (Fig. 6). Next, we found that
when expressed in Xenopus oocytes these truncated proteins
did not mediate a facilitated urea transport, in contrast to the
full-length wild type Jk+ protein, although all injected
cRNAs had a similar stability on a 3 days period, as seen by Northern
blot analysis. Further analysis revealed that the plasma membrane
fraction from oocytes expressing the functional urea transporter
(encoded by the Jk+ cRNA) carried a 46-69-kDa glycoprotein
component, which could be deglycosylated into a 36-kDa protein, as
expected for the Kidd/urea transporter protein (6, 8). On the contrary,
the Jk
(
6) and Jk
(
7) polypeptides were
neither detected in total cell lysates nor in the enriched plasma
membrane fraction from the oocytes, which could be explained by a rapid
intracellular degradation. Indeed, the predicted truncated proteins are
most likely misfolded by lack the transmembrane domains 3 and 4, and
transmembrane domain 5, including the hydrophilic loop carrying the
N-glycosylation site at Asn221, respectively.
Therefore, these findings provide a rationale explanation for the lack
of Kidd/urea transporter protein and defect in urea transport of
Jknull cells.
As the Kidd/HUT11 transcript is distributed widely in various organs (16), it is surprising that Jknull individuals who have a urea transport deficiency (35) did not suffer a clinical syndrome, except for a reduced capability to concentrate urine (36), as was the unexpected finding that donors of the blood group Colton null phenotype, who lack the water channel aquaporin-1, did not produce a severe or lethal phenotype (37, 38). It is postulated that mechanisms which compensate or reduplicate the function of the missing protein may exist. This should stimulate more studies of these transporters in red cell membranes and various organs. In addition, further investigations of other Jknull individuals from different ethnic groups (5), from both physological and fundamental aspects, may provide new information for addressing these issues.
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ACKNOWLEDGEMENTS |
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We are grateful to Phyllis S. Walker (Irwin Memorial Center, San Francisco, CA) for the help in collecting the B.S. blood sample and to Peter M. T Deen (University of Nijmegen, The Netherlands) for advice in oocyte methodology.
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FOOTNOTES |
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* This work was supported in part by INSERM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: INSERM U76, Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France. Tel.: (33) 1.44.49.30.00; Fax: (33) 1.43.06.50.19; E-mail: cartron{at}infobiogen.fr.
1 The abbreviations used are: PCR, polymerase chain reaction; RACE, 5'-rapid amplification of cDNA ends; UT, untranslated; SP, sense primer; AP, antisense primer; nt, nucleotide; bp, base pair(s); kb, kilobase pair(s).
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REFERENCES |
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