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TRANSFUSION MEDICINE
From INSERM U76, INTS, Paris, France;
Finnish Red Cross Blood Transfusion Service, Helsinki,
Finland; Auckland Institute of Technology, Auckland,
New Zealand; University of Texas-Houston Medical School,
Houston, TX.
Polymerase chain reaction genotyping of 32 unrelated
Jknull individuals originating predominantly from Polynesia
and Finland indicated that all were homozygous for the JK*B
polymorphism and that 17 of 32, including the 14 Polynesians, carried a
3'-acceptor splice site mutation of intron 5 that resulted in the
skipping of exon 6 (called mutation Jk The Kidd (JK) blood group antigens are carried by a
multispanning red blood cell (RBC) membrane glycoprotein of 45-kd
protein recently identified as the erythroid urea
transporter,1,2 although it is also expressed in the
kidney.3,4 The 2 major codominant alleles of the
JK gene, JK*A and JK*B, have a similar frequency in Caucasian populations (0.51 and 0.49, respectively)5,6 and define the 3 common phenotypes
Jk(a+b Many individuals of Asian and Polynesian extraction have been
identified as Jknull.8 Other populations
representing this rare phenotype include ethnic groups from
Brazil,9 India,10 and Japan.11
However, the Jknull phenotype is strikingly absent from
Caucasians although cases have been found in France,12 Australia,13 and Finland where it was first described in
1984.14
The Jknull phenotype is not associated with any obvious
clinical syndrome, except for a urine-concentrating
defect,15 which probably results from the absence of
Kidd/urea transporter protein on endothelial cell of the vasa recta in
the inner and outer medulla of the kidney.3,4 Although it
has been postulated that the erythrocyte Kidd/urea transporter might
prevent the swelling of RBCs when they leave the hyperosmotic
medulla,16 no hemolysis has been reported in
Jknull individuals. Jknull RBCs, however, are
easily identified by an increased resistance to lysis in aqueous 2 M
urea solution,17 a property widely used to screen for
individuals of Jknull phenotype.3,4
Recently, we have shown that the JK gene is composed
of 11 exons spreading over 30 kb of DNA19 and that the
JK*A/JK*B polymorphism resulted from a G838A transition,
causing a D280N amino acid substitution in the 3rd extracellular loop
of the Jk polypeptide.20 Moreover, 2 different splice-site
mutations were identified in 2 unrelated Jknull individuals
homozygous for the JK*B polymorphism. In the Chinese variant
B.S., the mutation affected the invariant G residue of the 3'-acceptor
splice site of intron 5, the Jk( In this report, we have further analyzed 32 samples of unrelated
Jknull donors originating mainly from Polynesia and Finland to determine their molecular basis. All Polynesian samples exhibited the Jk( Blood samples and reagents
Amplification by reverse transcription coupled with PCR
(RT-PCR)
Genomic DNA analysis by PCR The PCR genotyping assays were developed to detect the mutations that cause the basis of Jknull phenotypes. To identify the Jk( 6) variant, an allele-specific PCR reaction was performed between
primers SP-4, specific of the mutated allele, and AS-5 under stringent
conditions (94°C for 2 minutes [1 cycle]; 94°C for 30 seconds,
64°C for 30 seconds, 72°C for 30 seconds [30 cycles]; 72°C for
2 minutes [1 cycle]) using Taq polymerase. The 221-bp PCR product was
electrophoresed on a 1.8% (w/v) agarose gel stained with ethidium
bromide. To identify the Jk(7) variant, a PCR-restriction fragment
length polymorphism (PCR-RFLP) was carried out by a hemi-nested PCR
reaction (94°C for 2 minutes [1 cycle]; 94°C for 30 seconds, 54°C for 30 seconds, 72°C for 20 seconds [30 cycles]; 72°C for 2 minutes [1 cycle]) using the Expand High Fidelity system. The first
amplification was performed between primers SP-6 and AS-8 and the
second between primers SP-7 and AS-8 with 1/50 of the first reaction in
the same experimental conditions. The 76-bp PCR product was digested
with 5 U of Mse I restriction enzyme and electrophoresed on
a 15% acrylamide gel stained with ethidium bromide. To identify the
Finnish Jk(S291P) variants, a PCR-RFLP was performed between primers
SP-9 and AS-10 in the same conditions as above except that annealing
was performed at 58°C. The 81-bp PCR product was digested with 5 U of
Mnl I restriction enzyme and analyzed as previously on a
15% acrylamide gel. Each PCR reaction was performed in a total volume
of 50 µL containing 250 ng genomic DNA extracted from whole blood
cells using a Wizard Genomic DNA Purification kit from Promega
(Madison, WI). All primers used are given in Table 1.
Site directed mutagenesis of the Jk complementary DNA (cDNA) A nucleotide substitution A632T (resulting in a N211I substitution in the Jk polypeptide) was introduced into the wt Jk cDNA using a 2-step PCR approach with common SP-1 and AS-3 primers, in addition to sequence specific mutagenic primers overlapping each other, AS-11 and SP-12. Annealing was done at 60°C for the second PCR step. After subcloning into the EcoRV-digested pT7TS plasmid, the mutant construct was sequenced on both strands to confirm that the correct mutation has been obtained.Transcription-translation assays Polypeptides corresponding to wt Jk, Jk(S291P), and Jk(N211I) were in vitro synthesized from each pT7TS cDNA constructs (see below) using the transcription-translation coupled reticulocyte lysate kit from Promega in the presence of L-[35S]-methionine and with or without canine pancreatic microsomal membranes as recommended by the manufacturer. One tenth of the reaction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 15% separating gel on a discontinuous buffer system,22 followed by enlightening treatment (NEN, Boston, MA) and autoradiography.Oocyte urea flux measurements and immunocytochemistry After linearization of the pT7TS-wt Jk or Jk(S291P) 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 complementary RNAs (cRNAs) (0.1 ng/oocyte) in
collagenase-treated Xenopus laevi oocytes23 and
functional tests were realized 3 days after injection as described
previously.24 In parallel, groups of 3 to 6 oocytes
without chorionic membrane were embedded in paraffin as
described.25 Sections (7 µm thickness) were stained
overnight at 4°C with 10 µg/mL of affinity purified polyclonal
antibody directed against the N-terminal region (residues 8-22) of the Kidd/urea transporter protein, described
previously,1,3 and visualized with fluorescein-conjugated
goat antirabbit IgG (1:100 dilution) for 1 hour at room
temperature.26 Immunostaining of the oocyte plasma
membrane was imaged and quantified using a Nikon Eclipse TE300
microscope with epifluorescence illumination (Nikon, Paris, France)
coupled to a Biocom computer system of image integration (Visiolab 2000 program, Biocom, Les Ulis, France).
Northern blot analysis Total RNAs isolated from 6 injected oocytes, according to Chomezynski and Sacchi,27 were resolved by electrophoresis on 6% (w/v) formaldehyde, 1% (w/v) agarose gel, and transferred to nylon filters (Zeta-probe GT, Bio-Rad, Hercules, CA). Hybridization with a [32P]-labeled Jk cDNA probe (1.2 kb) using the random primed DNA labeling kit (Boehringer-Mannheim, Germany) was carried out at 65°C in 0.25 mol/L Na2HPO4, 7% (wt/vol) SDS. Stringent washes were performed in 0.02 mol/L Na2HPO4, 1% (wt/vol) SDS at 65°C for 30 minutes and exposed to Biomax-MR film with intensifying screen at80°C.
Cell culture, transfection, and flow cytometry Human erythroleukemic K562 cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown in Iscove's modified Dulbecco medium with Glutamax-1 (IMDM, Gibco BRL, Eragny, France) supplemented with penicillin-streptomycin and 10% fetal calf serum (FCS). To express wt Jk and Jk(S291P) polypeptides in K562 cells, wt Jk and Jk(S291P) cDNAs obtained by PCR between SP-1 and AS-3 primers (Table 1) were subcloned into pCEP4 episomal expression vector (Invitrogen, Leek, The Netherlands). After transfection using the Lipofectin-Reagent according to the manufacturer's instructions (Life Technologies, Gaithersburg, MD), stable K562 transfectants resistant to hygromycin (0.3 mg/mL) were selected for Jk protein expression by immunomagnetic separation using human anti-Jk3 antiserum and Biomag goat antihuman-IgG (Perseptive Biosystems, Connecticut, MA). The expression of Kidd/urea transporter polypeptides was examined by flow cytometry (FACScan, Becton Dickinson, San Jose, CA) using cultured cells (3-5 × 105) incubated 60 minutes at 22°C with human polyclonal or monoclonal anti-Jkb and anti-Jka at saturating concentration. The cells were then washed and stained with 100 µL of phycoerythrin (PE)-conjugated F(ab')2 fragments of goat antihuman IgG diluted 1:40 (Coulter/Immunotech, Marseille, France). After another washing step, 20 nmol/L of TO-PRO-1, molecular probes (Interchim, Monluçon, France), was added to the cell suspension 15 minutes before analysis, to exclude dead cells (TO-PRO positive cells).Western blot analysis The RBC membranes from donors of known phenotypes were prepared by hypotonic lysis.28 For Western blot analysis, RBC membrane proteins were separated by discontinuous SDS-PAGE, transferred to nitrocellulose sheets (Schleicher and Schull, Keene, NH; 0.1 µm) and incubated for 90 minutes at 37°C with 10 µg/mL of an affinity-purified rabbit antibody raised against the Kidd/urea transporter protein (see above). Bound antibodies were visualized with alkaline phosphatase-labeled goat antirabbit IgG 1:800 and the alkaline phosphatase substrate kit (BioRad).
Detection of mutations in JK gene from Jknull donors To determine the molecular defect occurring in Jknull phenotypes of 32 unrelated donors, among whom 14 originated from Polynesia, 15 from Finland, and 3 from diverse origins (Australia, Europe, and United States), we first used PCR-RFLP assays to screen for the JK*A/JK*B polymorphism20 and developed PCR genotyping assays for the 2 splice-site mutations previously identified in 2 Jknull individuals.19 To detect the Jk(6) mutation, an allele specific-PCR (AS-PCR) assay was performed
between the SP-4 primer, specific for the mutated JK*B
allele, and the common antisense AS-5 primer (Figure
1A). Homozygosity for this mutation was
determined by sequencing 5'-intron exon 6 boundaries as previously
described.19 In parallel, a hemi-nested PCR-RFLP assay was
developed to detect the Jk(7) mutation (Figure 1B). The results of
the genotyping are given in Table 2.
All 32 Jknull samples were found homozygous for the
JK*B polymorphism using the Mnl I PCR-RFLP previously
described20 (data not shown). A 221-bp AS-PCR product
characteristic of the Jk( Analysis of the Jk transcripts in Finnish Jknull donors Total reticulocyte RNA isolated from one Finnish Jknull sample was used as template to amplify by hemi-nested PCR the entire Jk cDNA using appropriate primer pairs as described in "Materials and methods." A 1.2-kb amplified fragment was readily amplified and sequence analysis revealed a T871C nucleotide transition that resulted in a S291P amino acid substitution located in a potential N-glycosylation motif (Figure 2A). Because the T871C substitution was correlated with the presence or the absence of an MnlI restriction site like the G838A transition, which caused the JK*A/JK*B polymorphism,20 a hemi-nested PCR-RFLP assay using primers SP-9 and AS-10 (Table 1) to test simultaneously the 2 positions was developed (Figure 2B). As expected, the 81-bp PCR product remained uncut in the Jk(ab+) control and
Jk(6) and Jk(7) mutants, whereas it was cleaved into 2 fragments
of 65 and 16 bp in the control Jk(a+b) sample. In contrast, 2 fragments of 69 and 12 bp were observed with 15 Finnish Jk(S291P)
mutants, demonstrating the homozygosity of the mutation in these donors
(Figure 2B, Table 2).
Because the Finnish samples carry a single missense mutation (S291P), we looked for a putative mutation in the promoter region of the JK gene. Amplification and sequencing of the proximal promoter (500 bp) upstream of the erythroid transcription initiation site of the JK gene did not reveal any alteration (data not shown). N-glycosylation of the Kidd/urea transporter protein In vitro transcription-translation coupled assays and ex vivo functional studies in Xenopus oocytes were performed to determine the effect of the missense mutation S291P on the expression level and functional properties of the Jk mutant polypeptide.In the cell-free transcription-translation coupled system, the mutant
plasmid pT7TS-Jk(S291P) directed the synthesis of a polypeptide of
apparent molecular mass of 36 kd with equal efficiency as the wild-type
pT7TS-Jk control plasmid (Figure 3). In
the presence of microsomal membranes, a minor band at 36 kd and a major
band at 40 kd corresponding to the glycosylated 36-kd product were detected with both plasmids, thus suggesting that the glycosylation pattern of the Jk(S291P) protein mutant was not affected. To
substantiate this point, we performed a site-directed mutagenesis of
asparagine 211 (N211I) in the consensus N-glycosylation
motif predicted to be exposed on the third extracellular loop of the Jk
polypeptide and which most likely is glycosylated.24 As
expected, the mutant plasmid pT7TS-Jk(N211I) directed only the
synthesis of the nonglycosylated 36-kd protein in the presence or
absence of microsomal membranes (Figure 3A). No protein material was
translated using the pT7TS plasmid alone.
Expression and functional analysis in Xenopus oocytes To test whether the Jk(S291P) mutant protein was functionally expressed as a urea transporter, the mutant cDNA carrying the T871C mutation was subcloned into the pT7TS vector and the cRNA transcript was synthetized and injected into Xenopus oocytes. The [14C]-urea uptake of Jk(S291P) cRNAs-injected oocytes was significantly increased when compared to water-injected control oocytes (Figure 4A), but was much lower when compared to the [14C]-urea uptake of wt Jk cRNA-injected oocytes (21.71 ± 0.43 versus 10.02 ± 1.66 pmol/oocyte at 1.5 minutes). The corresponding urea permeabilities calculated at 1.5 minutes were 53.0 ± 1.05 and 24.4 ± 4.05 × 10 6
cm/s, respectively, versus 0.63 ± 0.14 × 106
cm/s for water-injected control oocytes.
To test for a difference in stability between the wild-type and mutant injected cRNAs, total RNA was isolated from oocytes of the same batches and analyzed by Northern blot, which showed that similar levels of wild-type and mutant cRNAs were detectable 3 days after injection (Figure 4B). To localize the Jk(S291P) protein in oocytes, immunohistochemistry was performed on oocytes injected with water or cRNAs (Figure 4C). Staining with an affinity-purified antibody directed against the N-terminus of the Kidd/urea transporter protein revealed that the wt Jk protein was clearly expressed at the plasma membrane of oocytes, whereas oocytes expressing the mutant Jk(S291P) protein exhibited a weaker staining of the plasma membrane, with an increased cytoplasm reactivity (Figure 4C). Water-injected controls were negative, showing the specificity of antibody labeling. To provide a quantitative estimation of the plasma membrane staining of wild-type and mutant proteins, densitometric analysis of plasma membrane sections of 6 oocytes (4 fields analyzed per oocyte) for each type of cRNA injected was performed, based on the finding that the fluorescent signal is proportional to the amount of cRNA injected.29 Using the Visiolab 2000 program from Biocom, a 70% reduction of membrane insertion of the mutant protein was calculated, as the values for wild-type and mutant proteins were 187.63 ± 7.68 and 78.80 ± 1.58, respectively, versus 31.75 ± 0.64 for water-injected control oocytes (arbitrary units at magnification 20). Expression of the Jk(S291P) protein in human erythroleukemic K562 cells Expression vectors containing the wild-type and mutant cDNAs were transfected in K562 cells as described in "Materials and methods." After drug selection, flow cytometry analysis indicated that the human monoclonal (and human serum) anti-Jkb strongly reacted with K562 cells transfected with the wild-type but not the mutant cDNA construct (Figure 5). Anti-Jka (polyclonal or monoclonal) did not react with parental or transfected K562 cells (data not shown). The geometric mean ratios were 7.69 and 0.68 for K562 cells transfected with wild-type and mutant constructs (versus 0.74 for parental cells), respectively, indicating that only wild-type construct directed efficient Jk cell surface protein expression in an erythroid context.
Jk protein from common and Jknull RBCs Investigation by Western blot analysis indicated that the Jk protein was absent from RBC membrane preparations of all Jknull samples investigated (Figure 6). Indeed, the affinity-purified antibody that recognizes the N-terminus of the Jk protein reacted with a diffuse band of 46 to 69 kd present in membrane proteins from donors with a wt Jk phenotype, Jk(a+b), but did not react with membrane proteins from the
Jknull mutants Jk(6), Jk(7), and Jk(S291P).
Here we report the molecular basis of 32 unrelated
Jknull samples, most originating from Polynesia and
Finland. All were homozygous for the JK*B polymorphism.
These 2 countries have been selected for their genetic homogeneity and
a high incidence of the Jknull phenotype. Indeed, the
populations within Polynesia are known to be genetically remarkably
homogenous given the wide geographic region they
inhabit.30 This homogeneity may have been the result of an
initial founder effect, and later bottlenecks, coupled with an ongoing
network of contact that existed throughout Polynesia during and
immediately following settlement.31 Despite the
similarities between Polynesians, the incidence of some blood group
phenotypes showed differences in frequency between islands. Thus, the
ABO and Lewis phenotypes differed significantly32 as do
the frequencies of the Jknull phenotypes.33
This being so, our investigations showed that the 14 Jknull
donors from Polynesia resulted from the same mutation, Jk( The Jknull phenotype is relatively common in Finland, having a frequency of about 0.03%35 (and unpublished data) and an even distribution over the whole country. All 15 samples examined exhibited a T871C transition of the JK*B allele, resulting in the missense mutation S291P of the Kidd/urea transporter protein. Because this amino acid substitution was located in a NSS motif at position 289 to 291, it was predicted that this potential N-glycosylation site might be altered. The mere description of this mutation was described when this paper was under revision.36 However, neither functional nor glycosylation studies were carried out by the authors36 to relate the S291P mutation with the Jknull phenotype. Here, we have carried out such an analysis and at first we found that the wild-type and mutant Jk transcript typical of Finnish Jknull cells could be translated with an equal efficiency into major polypeptide species of 36 and 40 kd, as seen by transcription-translation analysis in the absence or in the presence of microsomal membranes, respectively. Further analysis by site-directed mutagenesis demonstrated that the asparagine at position 211 (located in the third predicted extracellular loop of the urea transporter24) of the wt Jk protein was the unique N-glycosylation site used. These findings indicate that the NSS motif at position 289 to 291 is not used as a glycosylation site, which correlates well with its predicted localization in the eighth transmembrane domain of the Jk polypeptide.24 Moreover, the complete absence of N-glycan on the mutant Jk(N211I) polypeptide indirectly demonstrates that the 3rd potential N-glycosylation site at position 125 to 127, which is located in the 3rd transmembrane domain of the Jk protein, is not used. Next, we found that the mutant Jk(S291P) protein behaves as a
functional urea transporter when expressed in Xenopus
oocytes, but the urea permeability of oocytes expressing this protein
was 50% lower than that of oocytes expressing the wt Jk protein,
although the 2 injected cRNAs were translated in vitro with equal
efficiency and had a similar stability for a 3-day period.
Immunostaining of paraffin-embedded sections of oocyte membranes
expressing the mutant Jk(S291P) and the wt Jk polypeptides, using an
affinity-purified antibody to the N-terminus of the Jk
protein, showed that there was less of the mutant protein than the wt
Jk protein at the cell surface and more intracellularly. Quantitative
staining analysis of oocyte membranes revealed that the reduced urea
permeability mediated by the mutant Jk(S291P) protein is consistent
with a reduced expression of the mutant protein at the oocyte plasma membrane, thus indicating that the specific urea transport activity of
the wild-type and mutant Jk protein is similar. The mutant protein
appears to be moving to the plasma membrane more slowly, presumably
because of the destabilizing S Oocyte studies clearly demonstrate that the missense mutation S291P
does not alter the urea transport properties of the mutant protein, but
they do not explain the absence of mutant protein in human RBCs. To
clarify this point cell surface expression of wild-type and mutant Jk
proteins was investigated in an erythroid context. Using episomal
expression vectors in K562 cells it was found that under forced
conditions of expression the wild-type but not the mutant Jk(S291P)
protein was efficiently expressed at the cell surface. Altogether,
these results strongly suggested that the presence of the S Our studies suggest that the oocyte expression system is more
permissive than the mammalian cell system because a fraction of the
mutant Jk protein could reach the cell surface and conferred urea
permeability to the injected oocytes. A similar phenomenon has been
shown for the common deleted form of the cystic fibrosis transmembrane
conductance regulator (CFTR- In conclusion, 3 different mutations of the JK gene
resulting in the Jknull phenotype have been currently
found, which all can be identified by PCR-genotyping assays. The
Jk(
Submitted August 31, 1999; accepted March 31, 2000.
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: Jean-Pierre Cartron, Unité INSERM U76, INTS, 6 rue Alexandre Cabanel, 75015 Paris, France; e-mail: cartron{at}infobiogen.fr.
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 N. Lucien, J. Chiaroni, J.-P. Cartron, and P. Bailly Partial deletion in the JK locus causing a Jknull phenotype Blood, February 1, 2002; 99(3): 1079 - 1081. [Abstract] [Full Text] [PDF]
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 R. T. Timmer, J. D. Klein, S. M. Bagnasco, J. J. Doran, J. W. Verlander, R. B. Gunn, and J. M. Sands Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1318 - C1325. [Abstract] [Full Text] [PDF]
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
P
mutation. After transfection in erythroleukemia K562 cells the
wild-type, but not the mutant, protein was efficiently expressed at the
cell surface. Because the Jk(S291P) mutant polypeptide was not present
in human red cells from Jknull individuals, expression data
in the erythroid context clearly indicates that the
S
