The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders whose hallmarks are ineffective erythropoiesis, hemolysis, and morphological abnormalities of erythroblasts in ...bone marrow. We have identified a missense mutation in
KLF1 of patients with a hitherto unclassified CDA. KLF1 is an erythroid transcription factor, and extensive studies in mouse models have shown that it plays a critical role in the expression of globin genes, but also in the expression of a wide spectrum of genes potentially essential for erythropoiesis. The unique features of this CDA confirm the key role of KLF1 during human erythroid differentiation. Furthermore, we show that the mutation has a dominant-negative effect on KLF1 transcriptional activity and unexpectedly abolishes the expression of the water channel AQP1 and the adhesion molecule CD44. Thus, the study of this disease-causing mutation in
KLF1 provides further insights into the roles of this transcription factor during erythropoiesis in humans.
The Kidd (JK) blood group locus encodes the urea transporter hUT-B1, which is expressed on human red blood cells and other tissues. The common JK*A/JK*B blood group polymorphism is caused by a single ...nucleotide transition G838A changing Asp-280 to Asn-280 on the polypeptide, and transfection of erythroleukemic K562 cells with hUT-B1 cDNAs carrying either the G838 or the A838 nucleotide substitutions resulted in the isolation of stable clones that expressed the Jka or Jkb antigens, respectively, thus providing the first direct demonstration that the hUT-B1 gene encodes the Kidd blood group antigens. In addition, immunochemical analysis of red blood cells demonstrated that hUT-B1 also exhibits ABO determinants attached to the single N-linked sugar chain at Asn-211. Moreover, immunoadsorption studies, using inside-out and right-side-out red cell membrane vesicles as competing antigen, demonstrated that the C- and N-terminal ends of hUT-B1 are oriented intracellularly. Mutagenesis and functional studies by expression in Xenopus oocytes revealed that both cysteines Cys-25 and Cys-30 (but not alone) are essential for plasma membrane addressing. Conversely, the transport function was not affected by the JK*A/JK*Bpolymorphism, C-terminal deletion (residues 360–389), or mutation of the extracellular N-glycosylation consensus site and remains poorly para-chloromercuribenzene sulfonate (pCMBS)-sensitive. However, transport studies by stopped flow light scattering using Jk-K562 transfectants demonstrated that the hUT-B1-mediated urea transport is pCMBS-sensitive in an erythroid context, as reported previously for the transporter of human red blood cells. Mutagenesis analysis also indicated that Cys-151 and Cys-236, at least alone, are not involved in pCMBS inhibition. Altogether, these antigenic, topologic, and functional properties might have implications into the physiology of hUT-B1 and other members of the urea transporter family.
Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol ...facilitator of Escherichia coli, are dependent upon the analytical technique used. Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea. At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells. Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems:Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3. By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono- and dimeric species in conditions under which AQP1 preserved its tetrameric structure. Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure. Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one.
The Kidd (JK) blood group locus encodes a urea transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human ...erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 urea transporter, except for a Lys44→ Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/urea transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels inXenopus oocytes, the physiological Kidd/urea transporter HUT11A conferred a rapid transfer of urea (which was insensitive top-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the urea transport was poorly inhibited byp-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A transporter confers urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell urea transporter when expressed at unphysiological high levels.
Here, we report the biochemical and genetic basis of the Vel blood group antigen, which has been a vexing mystery for decades, especially as anti‐Vel regularly causes severe haemolytic transfusion ...reactions. The protein carrying the Vel blood group antigen was biochemically purified from red blood cell membranes. Mass spectrometry‐based de novo peptide sequencing identified this protein to be small integral membrane protein 1 (SMIM1), a previously uncharacterized single‐pass membrane protein. Expression of SMIM1 cDNA in Vel− cultured cells generated anti‐Vel cell surface reactivity, confirming that SMIM1 encoded the Vel blood group antigen. A cohort of 70 Vel− individuals was found to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence of SMIM1. The genetic homogeneity of the Vel− blood type, likely having a common origin, facilitated the development of two highly specific DNA‐based tests for rapid Vel genotyping, which can be easily integrated into blood group genotyping platforms. These results answer a 60‐year‐old riddle and provide tools of immediate assistance to all clinicians involved in the care of Vel− patients.
The study deciphers the molecular basis of the Vel‐ blood group, a rare yet important antigen responsible for life‐threatening transfusion accidents.
BACKGROUND: The Colton blood group system currently comprises three antigens, Coa, Cob, and Co3. The latter is only absent in the extremely rare individuals of the Colton “null” phenotype, usually ...referred to as Co(a–b–), which lack the water channel AQP1 that carries the Colton antigens. The discovery of a Co(a–b–) individual with no AQP1 deficiency suggested another molecular basis for the Co(a–b–) phenotype.
STUDY DESIGN AND METHODS: Red blood cells were analyzed by stopped‐flow light scattering and Western blotting and typed by hemagglutination and flow cytometry. Genotyping by sequencing and polymerase chain reaction–restriction fragment length polymorphism was applied. An expression system for Colton antigens was developed in mammalian cells.
RESULTS: Although Co(a–b–), the proband expressed fully functional AQP1 and had developed a novel Colton alloantibody. Sequencing of AQP1 revealed a homozygous nucleotide change (140A>G) encoding the single‐amino‐acid substitution Q47R. A second case was identified due to the presence of this novel Colton alloantibody. By generating an expression system for Colton antigens in K‐562 cells, the Q47R substitution was shown to inhibit the expression of both Coa and Cob antigens. Other naturally occurring single‐amino‐acid substitutions, that is, A45T, P38L, and N192K, were also studied in this Colton antigen expression system.
CONCLUSIONS: The Co(a–b–) phenotype can be generated by a functional AQP1 allele, that is, AQP1 140G encoding AQP1 (Q47R) and allowing the development of a novel Colton alloantibody. This study also shows that the Cob antigen can be produced by at least two different substitutions at Amino Acid Position 45, that is, A45V and A45T.
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-specificcis-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 Xenopusoocytes 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.
BACKGROUND: McLeod syndrome is a rare X‐linked neuroacanthocytosis syndrome with hematologic, muscular, and neurologic manifestations. McLeod syndrome is caused by mutations in the XK gene whose ...product is expressed at the red blood cell (RBC) surface but whose function is currently unknown. A variety of XK mutations has been reported but no clear phenotype‐genotype correlation has been found, especially for the point mutations affecting splicing sites.
STUDY DESIGN AND METHODS: A man suspected of neuroacanthocytosis was evaluated by neurologic examination, electromyography, muscle biopsy, muscle computed tomography, and cerebral magnetic resonance imaging. The McLeod RBC phenotype was disclosed by blood smear and immunohematology analyses and then confirmed at the biochemical level by Western blot analysis. The responsible XK mutation was characterized at the mRNA level by reverse transcription–polymerase chain reaction (PCR), identified by genomic DNA sequencing, and verified by allele‐specific PCR.
RESULTS: A novel XK splice site mutation (IVS1‐1G>A) has been identified in a McLeod patient who has developed hematologic, neuromuscular, and neurologic symptoms. This is the first reported example of a XK point mutation affecting the 3′ acceptor splice site of Intron 1, and it was demonstrated that this mutation indeed induces aberrant splicing of XK RNA and lack of XK protein at the RBC membrane.
CONCLUSION: The detailed characterization at the molecular biology level of this novel XK splice site mutation associated with the clinical description of the patient contributes to a better understanding of the phenotype‐genotype correlation in the McLeod syndrome.
A new alteration of the blood group JK*A allele was identified in a Jknull patient from Tunisia with an allo–anti-Jk3 in her serum. Southern blot and exon mapping analyses revealed an internal ...deletion within the Kidd (JK) locus encompassing exons 4 and 5. Sequence analysis of the Jk transcript showed that exons 4 and 5 were missing but were replaced by a 136–base-pair (bp) intron 3 sequence located 315 bp and 179 bp upstream from exon 4. This sequence is flanked by typical donor–acceptor cryptic splice sites used in the mutant but not in the normal JK gene. Because the translation initiation codon is located in exon 4, the Jk protein is not produced.
A new alteration of the blood group JK*A allele was identified in a Jk(null) patient from Tunisia with an allo-anti-Jk3 in her serum. Southern blot and exon mapping analyses revealed an internal ...deletion within the Kidd (JK) locus encompassing exons 4 and 5. Sequence analysis of the Jk transcript showed that exons 4 and 5 were missing but were replaced by a 136-base-pair (bp) intron 3 sequence located 315 bp and 179 bp upstream from exon 4. This sequence is flanked by typical donor-acceptor cryptic splice sites used in the mutant but not in the normal JK gene. Because the translation initiation codon is located in exon 4, the Jk protein is not produced.
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