|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2147-2156
Arg89Cys Substitution Results in Very Low Membrane Expression of the
Duffy Antigen/Receptor for Chemokines in Fyx Individuals
By
Christophe Tournamille,
Caroline Le Van Kim,
Pierre Gane,
Pierre
Yves Le Pennec,
Francis Roubinet,
Jérôme Babinet,
Jean
Pierre Cartron, and
Yves Colin
From INSERM U76, Institut National de la Transfusion Sanguine, Paris,
France; the Centre National de Référence sur les Groupes
sanguins, Paris, France; the Centre Régional de Transfusion
Sanguine, Toulouse, France; and the Etablissement de Transfusion
Sanguine, Hopital Pitié Salpetrière, Paris, France.
 |
ABSTRACT |
The Duffy (FY) blood group antigens are carried by the DARC
glycoprotein, a widely expressed chemokine receptor. The molecular basis of the Fya/Fyb and Fy(a-b-) polymorphisms
has been clarified, but little is known about the Fyx
antigen and the FY*X allele associated with weak expression of Fyb, Fy3, Fy5, and Fy6 antigens. We analyzed here the
structure and expression of the FY gene in 4 Fy(a-bweak) individuals. As compared with Fy(a-b+)
controls, the Fy(a-bweak) red blood cell membranes
contained residual amount of DARC polypeptide and these cells were
poorly bound by anti-Fy antibodies and chemokines. The FY gene
from Fy(a-b+) and Fy(a-bweak) individuals differed by one
substitution, C286T. The resulting Arg89Cys amino acid change reduced
the binding of anti-Fy antibodies and chemokines to DARC transfectants.
We concluded that the Fybweak donors carried the
FY*X allele at the FY locus and that the Fyx
antigen corresponds to highly reduced expression of a grossly normal
Fyb polypeptide caused by the Arg89Cys substitution.
Because FY is a single copy gene, this defect should also
affect DARC expression in nonerythroid cells. Because the
Fyx phenotype is not associated with apparent clinical
consequences, we discussed these findings in the light of the putative
roles of DARC in various tissues. Finally, we developed a
Fyx DNA typing assay that should be useful for genetic
studies and clinical transfusion medicine.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THE DUFFY BLOOD group antigens are of
wide interest in clinical medicine because of their involvement in
transfusion incompatibilities and hemolityc disease of the newborn
(HDN).1 The Duffy blood group system should represent one
of the best illustrations of the link between genetics and biology.
Indeed, before their molecular cloning, the Duffy antigens were already
recognized as the erythrocyte receptor for malaria parasites and for
chemokines because Duffy-positive but not Duffy-negative erythrocytes
can be invaded by Plasmodium vivax2 and
Plasmodium knowlesi3 and can bind interleukin-8
(IL-8).4,5 The Duffy antigens are carried by a 336 amino
acid glycoprotein, originally named gpD,6 that exhibits
significant protein sequence homology with the human and rabbit IL-8
receptors and that is most likely organized into seven transmembrane
domains, like all members of the G-protein-coupled chemokine
receptors.7 gpD is now referred to as the promiscuous chemokine receptor or the Duffy antigen/receptor for chemokine (DARC),
because it expresses all Fya/b, Fy3, and Fy6
antigens7-9 and can bind chemokines of both the CC (RANTES,
MCP-1) and CXC (IL-8, MGSA) classes.7,8 In addition to
erythroid cells, DARC is expressed on endothelial cells lining postcapillary venules throughout the body,10,11 on vascular endothelial and epithelial cells in some nonerythroid
organs,12 and on Purkinje cells in the
cerebellum.13 The same DARC polypeptide isoform is
expressed in all tissues studied so far,10,12,14 including
the brain, where a larger FY mRNA (7.5 kb v 1.35 kb) is
produced by the use of a specific promoter.14 The
function(s) of the Duffy antigens as a widely expressed promiscuous
chemokine receptor is not elucidated. However, it has been postulated
that DARC on red blood cells (RBCs) could act as a sink or scavenger to
inactivate excess chemokines released into the
circulation.4 Accordingly, it has been demonstrated that
IL-8 released after myocardial infarction is mainly bound to
erythrocytes.15 There is no evidence that DARC on
nonerythroid cells could transduce a signal across the membrane upon
chemokine binding.7 However, transfectant cell analysis
indicated that endothelial DARC could internalize
ligands,11 and a recent study suggested that DARC might
participate to transcytosis and surface presentation of IL-8 by venular
endothelial cells,16 an important site for
chemokine-induced leukocyte emigration during inflammatory process.
The receptor properties of DARC for malaria parasites and chemokines,
together with the existence of phenotypes associated with quantitative
and/or qualitative alteration of Fy antigen expression,
provided new interest in the elucidation of the molecular genetic basis
of the Duffy blood group system. The single copy FY gene is
composed of two exons,17 and the two common alleles in
Caucasians, FY*A and FY*B, differ by a single G to A
nucleotide substitution resulting in the amino acid change Gly42Asp in
the NH2 extracellular domain.9,18,19,20 In Caucasian
populations, the three phenotypes Fy(a+b-), Fy(a-b+), and Fy(a+b+)
given by these codominant alleles have a frequency of 0.195, 0.33, and 0.475, respectively. The phenotype Fy(a-b-), conveyed by homozygosy for
the FY*Fy allele, is extremely rare in Caucasians but
represents the major phenotype in blacks. The FY*Fy allele
corresponds to a normal FY*B coding sequence but exhibits a
mutation in the promoter region, T-46C, that, by disrupting a binding
site for the h-GATA-1 erythroid transcription factor, abolishes
expression of the FY gene in erythroid but not in nonerythroid
cells.21,22 Thus, Fy(a-b-) individuals resist
Plasmodium vivax infection because they lack DARC on their
RBCs18 but have no obvious abnormality in the regulation of
inflamation most likely because they express DARC normally on their
nonerythroid tissues.11,12
In 1965, Chown et al23 reported that the very weak and
variable reaction of some RBCs with anti-Fyb antibodies
accounts for many seeming anomalies of inheritance in Caucasian
families. These investigators postulated that the Fybweak
phenotype is conveyed by a fourth allele at the FY locus with a
frequency of 2%, which they named FY*X because the total gene product was not certain. Further serological studies indicated that expression of Fy3, Fy5, and Fy6 antigens is also depressed in
Fybweak RBCs from donors with the putative genotype
FY*X/*X.24- 28 Although it has been proposed that
Fyx should correspond to a quantitative rather than a
qualitative variant of Fyb, the molecular nature, the
biological properties, and the genetic background of the
Fyx antigen and of the FY*X allele, constituted one
of the dark holes in the study of the Duffy blood group system. To
clarify these issues, we analyzed the structure and expression of the
FY locus from 4 Fy(a-bweak) donors. We identified a
mutation that accounts for very low DARC expression and chemokine
binding to RBCs and transfected eukaryotic cells. Our results, together
with the previous characterization of FY*A, FY*B, and
FY*Fy, provide the definitive proof that the Duffy blood group
system is controlled by four alleles.
| |
MATERIALS AND METHODS |
Blood samples.
Blood samples from healthy donors were collected on EDTA and the Fy
phenotypes were determined by agglutination studies using the
antiglobulin gel test (Diamed SA, Morat, Switzerland) and by subsequent
adsorption/elution studies, when necessary. The Fy(a-bweak)
donors BE.T (and family members) and donors SEV and BAR were obtained
from the Etablissement de Transfusion Sanguine of Hopital Pitié
Salepêtrière (Paris, France) and the Centre National de
Réference pour les Groupes Sanguins (CNRGS; Paris, France), respectively. The Fy(a-bweak) blood sample TAR was
collected at the Centre Regional de Transfusion Sanguine of Toulouse
(Toulouse, France).
Materials.
125I-labeled IL-8, MGSA, and RANTES (specific activity,
2,200 Ci/mmol) were obtained from DuPont NEN (Boston, MA). Unlabeled and fluorescent fluorescein isothiocyanate
(FITC)-conjugated recombinant IL-829 were
donated by Dr A. Proudfoot (Glaxo Wellcome Research and Development,
Geneva, Switzerland). The anti-Fy6 (i3A), anti-Fy3 (CRC-512), and
anti-Fya MoAbs were kindly provided by Dr D. Blanchard
(CRTS, Nantes, France), Dr Makato Uchikawa (Japanese Red Cross, Tokyo,
Japan), and Dr F. Buffiere (ETS, Bordeaux, France), respectively.
Anti-Fya and anti-Fyb human polyclonal
antibodies were from Ortho Diagnostic Systems (Raritan, NJ). The
anti-p55 rabbit polyclonal antibody, prepared by immunizing rabbit with
synthetic peptide residues 28 to 47 of the human p55
protein,30 was donated by Dr P. Bailly (INTS, Paris,
France).
Polymerase chain reaction (PCR) amplification of FY gene and
transcripts.
Total RNAs were extracted from 400 µL of whole peripheral blood by
the miniscale acid-phenol-guanidium method.31 The pellet was resuspended in 50 µL H2O, and 10 µL was used to
produce first cDNA strands using the first-strand cDNA synthesis kit
(Pharmacia, Uppsala, Sweden). One sixth of the cDNA products were
enzymatically amplified between primers FY100,
5 -GAACCAAACGGTGCCATGGGGAACTGTCTG-3 (sense, positions  15 to +15),
and FY99, 5- GGGAAGAGAACTAGGATTTGCTTCCAAGGG-3 (antisense, positions
+1021 to +992). Genomic DNA (200 ng) isolated from whole blood was
amplified between primers FY94, 5-AACAGCGTCCCCTAACCAG-3 (sense,
positions 1253 to 1236), and FY99. Thirty cycles of PCR were
performed as follows: 1 minute at 94°C, 1 minute at 58°C, and 2 minutes at 72°C. PCR products were subcloned in plasmid vector and sequenced by the dideoxy chain termination method
using an ALFexpress automatic DNA sequencer (Pharmacia).
Restriction analysis of PCR-amplified genomic sequences.
For the FY*X restriction fragment length polymorphism
(RFLP) assay, the PCR reactions were performed with 200 ng
of leukocyte DNA between primers FY7, 5-ACTCTGCACTGCCCTTCTTC-3
(sense, positions +176 to +195), and FY57, 5-TGGGCAAAGGCTGAGCCA-3
(antisense, positions +428 to +411), under the following conditions: 30 cycles of 40 seconds at 94°C, 1 minute at 58°C, and 45 seconds
at 72°C. PCR products were purified on Ultrafree-MC (30,000) filter
units (Millipore, Bedford, MA) and one third was digested for 2 hours with 20 U of the Aci I restriction enzyme. Restriction
fragments were directly analyzed in 12% acrylamide minigels.
FY*A/FY*B genotyping was performed as decribed.9
FY*Fy typing was performed essentially as
described,21 except that the reverse primer P39 was changed to FY97, 5 TGTGGCAGACAGTTCCCCATGG-3 (position +40 to +27), to better
separate the FY*Fy specific Sty I restriction fragment (64 bp) generated by the T-46C mutation from other fragments.
Construction of Fy expression plasmid and mutagenesis.
pcDNA-3 expression vector (Invitrogen BV, Leek, The
Netherlands) carrying the coding sequence of the major isoform
(spliced) of Fyb was described elsewhere.32
Mutagenesis was subsequently performed on the recombinant plasmids by
the use of PCR primers carrying appropriate nucleotide substitutions
and the Quick Change Site Directed Mutagenesis Kit (Stratagene, La
Jolla, CA). Inserts of the mutated plasmids were sequenced as described
above.
Cell culture and transfection.
COS-7 cells were obtained from the American Type Culture Collection
(Rockville, MD) and were grown in Iscove medium supplemented with 10%
fetal calf serum and 50 µg/mL penicillin and streptomycin. Cells (3 × 106/assay) were transfected with 10 µg of
recombinant plasmid using Lipofectin reagent (Life Technologies, SARL,
Cergy-Pontoise, France). Transiently transfected COS-7
cells were analyzed after 36 hours of culture.
Flow cytometry analysis.
Expression of Duffy antigens on RBCs or transfectant cell lines was
measured on a FACScan flow cytometer (Becton Dickinson, San Jose, CA)
using anti-Fya, anti-Fy6, and anti-Fy3 MoAbs and
anti-Fyb PoAb. Cells (3 × 105) were
incubated for 60 minutes at 22°C with appropriate dilution of
antibody in 0.15 mol/L phosphate-buffered saline (PBS). After washing
with PBS supplemented with 0.5% bovine serum albumin
(BSA), the cell suspension was incubated with
fluorescein-conjugated antimouse or antihuman IgG (H+L) (Immunotech,
Marseille, France). After another washing step, 0.1 ng of propidium
iodide (PI) was finally added to 1 mL of cell suspension. PI-positive
cells (dead cells) were excluded from analysis. Fy(a-b-) RBCs or
mock-transfected cells and irrelevant mouse and human MoAbs were used
as negative controls.
Receptor binding assay.
RBCs (108) and COS-7 cell transfectants (106)
were analyzed for their ability to bind 125I-chemokines as
previously described.32 Cells transfected with the pcDNA3
vector alone were used as negative controls.
The binding of FITC-conjugated IL-8 to RBCs was directly measured by
flow cytometry analysis.
Protein chemistry.
RBCs membranes from Fy-typed donors were prepared by hypotonic
lysis.33 For Western blot analysis, RBC membrane proteins (50 µg) were separated by discontinuous sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)34
using a Novex apparatus (San Francisco, CA) and transferred to
nitrocellulose sheets. The blot was incubated with an appropriate
dilution of the i3A anti-Fy 6 MoAb and then with antimouse IgG
peroxydase-tagged antibody (Biosis, Compiègne,
France). Finally, the immunoblot was stained with the ECL
chemiluminescent system from Amersham (Bucks, UK) and exposed to x-ray
film.
| |
RESULTS |
Duffy antigen expression on Fy(a-bweak) RBCs.
In agglutination studies with polyclonal anti-Fy reagents, RBCs from
donors BAR and SEV were unreactive with the anti-Fya
antibodies and gave positive reactions with some anti-Fyb
antisera, but with an agglutination titer fivefold lower than those
given by Fy(ab+) and Fy(a+b+) controls. RBCs from donors TAR and
BE.T were unreactive with all anti-Fya and
anti-Fyb reagents and thus were initially considered as
Fy(ab) (data not shown). However, in subsequent
adsorption-elution experiments, these two RBC samples yielded an
antibody with Fyb specificity. From these analysis, donors
BAR, SEV, TAR, and BE.T were phenotyped Fy(abweak). The
altered expression of Fyb on these RBC samples was better
shown by flow cytometry analysis (Table 1).
When compared with the Fy(ab+), Fy(a+b+), Fy(a+b), and
Fy(ab) controls, a faint fluorescence signal was detected on the
Fybweak RBCs, ranging from 3% to 7.7% of that obtained
with Fy(ab+) (FY*B/*B) control RBCs. Similarly,
the use of anti-Fy3 and anti-Fy6 monoclonal antibodies (MoAbs) also
showed a drastic alteration of Fy3 and Fy6 antigen expression at the
surface of the Fy(abweak) RBCs. The decrease of anti-Fy3
and anti-Fy6 antibody binding capacity were strictly correlated between
samples and the RBCs from donor BAR were stained approximatively twice
as much as the RBCs from the 3 other Fy(abweak) donors
with all anti-Fy antibodies (Table 1). The apparent Duffy site numbers
per cell, as estimated from the anti-Fy3 and anti-Fy6 MoAb binding
capacity, were 2,200 to 3,400 for the Fy(ab+) controls; 150 to 230 for SEV, TAR, and BE.T; and 250 to 470 for BAR. As expected,
anti-Fya MoAb bound neither to Fy(abweak)
nor to the Fya-negative control RBCs. The
Fybweak, Fy3weak, and Fy6weak
phenotype of the 4 Fy(abweak) donors strongly suggested
that they belong to the Fyx phenotype originally described
and therefore should carry the FY*X allele of the Duffy blood
group system.
Chemokine binding to Fy(abweak) RBCs.
The Duffy antigens expressed on the Fy(abweak) RBCs were
further characterized by analyzing their chemokine receptor properties. In a first set of experiments, the binding of IL-8 to the four Fy(abweak) samples was measured by flow cytometry using
increasing concentrations of fluorescent FITC-conjugated IL-8
(Fig 1). As expected, IL-8 bound strongly
to Fy(ab+) RBCs but did not bind significantly to Fy(ab) RBCs
that are deficient in DARC glycoprotein.6 Fy(abweak) RBCs exhibited little but reproducible
binding to IL-8 as compared with Fy(ab) RBCs, with the staining
of BAR RBCs being twice as strong as compared with the other
Fy(abweak) samples. In a second set of experiments, the
binding of 125I-labeled chemokines to
Fy(abweak) samples BAR and SEV was examined.
Quantitative analysis indicated that IL-8 binding to these cells
represented 24% and 12%, respectively, of the binding to the positive
controls (Table 2). When tested for binding
of 125I-labeled MGSA and RANTES, the RBCs from BAR also
exhibited a 60% and 80% reduction of chemokine binding, respectively,
as compared with Fyb-positive controls.
View larger version (17K):
[in this window]
[in a new window]
| Fig 1.
Flow cytometry analysis of IL-8 binding to RBCs. RBCs (5 × 104) from the Fy(abweak) donors SEV and
BAR and from Fy(ab+) and Fy(ab) controls with the determined
genotypes FY*B/*B and FY*Fy/*Fy, respectively, were
incubated with increasing concentration of fluorescent FITC-conjugated
IL-8. Chemokine binding was analyzed by flow cytometry. Results are
expressed as the specific IL-8 binding (arbitrary units) versus ligand
concentration. Nonspecific signal was determined when incubation with
the FITC-labeled IL-8 was performed in presence of a 100-fold excess of
unlabeled IL-8.
|
|
Western blot analysis.
The amount of DARC polypeptide in erythrocyte membranes from donors
with different Fy phenotypes was compared by Western blot analysis,
using the iA3 MoAb directed against the Fy6 antigen (Fig 2). As expected, no signal was
observed in the Fy(ab) negative controls. DARC was consistently
detected in membranes from Fy(a+b), Fy(a+b+), and Fy(ab+)
controls with the FY*A/*A, FY*A/*B, and FY*B/*B
genotypes, respectively, whereas a signal 50% lower was observed in
membranes from donors with the FY*A/*Bweak
(BE.C) and FY*Fy/*A (BE.L) genotypes, respectively (see
below). Nevertheless, DARC was not detected in membranes from SEV,
BE.T, and BAR Fy(abweak) donors (Fig 2A). However,
prolonged radiographic exposure showed that these membranes had a
barely detectable signal, equivalent to less than 5% of the normal
intensity, with the signal in SEV and BE.T samples being 50% of that
in BAR membranes (Fig 2B). As a control, all samples exhibited normal
amounts of the p55 peripheral protein (Fig 2C).
View larger version (57K):
[in this window]
[in a new window]
| Fig 2.
Immunoblot analysis of RBC membrane proteins. Total
membrane proteins (60 µg) separated on SDS-PAGE were blotted on
nitrocellulose sheets and incubated with the murine anti-Fy6 MoAb i3A
and with a rabbit anti-p55 antibody. The DARC glycoproteins and p55
proteins were visualized by chemioluminescence autoradiography using
goat antimouse or goat antirabbit IgG conjugated to horseradish
peroxidase, as second antibodies, respectively. The 35- to 45-kD DARC
glycoprotein forms aggregates migrating as bands of 90 and 200 kD.45
|
|
FY gene and transcript analysis.
RNAs extracted from whole blood of the 4 Fy(abweak)
donors and from a Fy(ab+) control were reverse transcribed to cDNA
and used as template to amplify the entire coding region for the major (spliced) isoform of DARC between primers FY100 and FY99. A PCR product of the expected size (1,036 bp) was obtained in all samples (data not shown) and subcloned in plasmid vector. Sequence analysis of
several clones from each Fy(abweak) sample showed one
kind of cDNA that differed from the common FY*B allele by only
one substitution, C286T (+1 taken as the erythroid cap
site21; Fig 3). A survey of the
literature indicated that T286 has never been found in the
FY*A, FY*B, or FY*Fy alleles of donors with common Duffy phenoypes. The C286T nucleotide change resulted in the
amino-acid substitution Arg to Cys at position 89 of the DARC polypeptide. It is noteworthy that all Fybweak cDNA clones
carried a G at nucleotide 319, whereas G or A, resulting in Ala100Thr
substitution, was identified at this position by sequencing the
FY*B or FY*A alleles from Fy(ab+) or Fy(a+b+) donors7,20 (and our unpublished data).
View larger version (10K):
[in this window]
[in a new window]
| Fig 3.
Schematic comparative structure of the DARC cDNAs isolated
from whole blood of Fy(ab+) and Fy(abweak) donors.
The full-length major isoform (spliced) of the FY transcripts was
isolated by RT-PCR. The nucleotide sequences of Fy(ab+) and
Fy(abweak) cDNA clones were identical, except for the
C286T substitution (+1 taken as the erythroid cap site) resulting in
the Arg89Cys polymorphism on the DARC protein. G was found at
nucleotide position 319 in all clones from four unrelated
Fy(abweak) donors, whereas G or A, resulting in
Ala100Thr amino acid change, could be found in the FY*B allele
from Fy(a+b+) and Fy(a-b+) donors. The nucleotide residue found
at the Fya/Fyb-associated polymorphic position
(G125A) is indicated.
|
|
The C286T nucleotide substitution was confirmed at the genomic level
after amplification of the FY gene, including intron 1 and
1,253 nucleotides in 5 of the erythroid cap site. However, in contrast
to the cDNA analysis, sequence analysis of several genomic clones
suggested homozygosity for donor BAR and showed the heterozygosity of
donors SEV, BE.T, and TAR. One species of clones from SEV, BE.T, and
TAR samples carried a normal Fyb coding sequence and
exhibited the T-46C mutation in the promoter region typical of
FY*Fy and was previously shown to abolish the erythroid
expression of this bone marrow silent FY*B
allele.21 Accordingly, this allele could not be shown by
cDNA analysis of erythroid cells. Conversely, the second
species of clones exhibited the C286T substitution but no additional
polymorphism compared with the noncoding and 5 flanking sequences of
FY*B gene. These results indicated that the genotype of the
Fy(a-bweak) donors SEV, TAR, and BE.T was FY*Fy/*X
and suggested that the genotype of donor BAR was FY*X/*X.
Fyx DNA typing by PCR-RFLP.
The substitution identified at nucleotide 286 of the FY mRNAs was
correlated with the presence or the absence of an Aci I restriction site (CCGC CTGC) on the Fy(ab+) and
Fy(abweak) clones, respectively. To confirm that the
C286T substitution is associated with the Fybweak
phenotype, a PCR-RFLP assay was developed. Primers FY7 and FY57 (see
Materials and Methods) were designed to amplify a 251-bp fragment
encompassing this polymorphic position on the FY gene. PCR-RFLP
of genomic DNAs from Fy typed donors have been performed and typical
results are shown in Fig 4. After
Aci I digestion, the 251-bp PCR product was cleaved in two
fragments of 88 and 163 bp in all Fy samples except those with the
Fy(abweak) or Fy(a+bweak) phenotypes. Only
the uncleaved 251-bp fragment was observed in donor BAR, whereas the
251-, 163-, and 88-bp fragments were all detected in the heterozygous
Fy(a+bweak) sample (BE.C) as well as in samples BE.T, SEV,
and TAR.
View larger version (42K):
[in this window]
[in a new window]
| Fig 4.
Full Duffy DNA typing. (A) Strategy of the PCR-RFLP for the
detection of the C286T substitution specific of FY*X allele.
Primers Fy7 and Fy57 were designed to amplify a 251-bp FY
fragment encompassing the single base substitution identified between
the Fy(ab+) and Fy(abweak) clones and that was
correlated with an allele-specific Aci I restriction site.
Nucleotide numbers are as in Fig 3 and do not take into account the
intronic sequence of the FY gene. (B) DNA from donors with the
indicated Duffy phenotype was used as templates in PCR-RFLP assays. Ten
donors with each control phenotypes were analyzed and typical results
are shown. The detection of the FY*A-, FY*B-, and
FY*Fy-associated polymorphisms (G125A and C-46T, respectively)
were based on Ban I and Sty I RFLP, as previously
described, with some modifications for FY*Fy typing (see
Materials and Methods). The TAR sample gave the same RFLP pattern as
SEV and BE.T. The tree of the BE. family is shown to follow the
inheritance of the FY*X mutation and to demonstrate that the
presence at the heterozygous state of the silent allele FY*Fy
in BE. father accounts for the apparent exclusion of paternity (BE.T
being Fya-negative, with both parents being
Fya-positive).
|
|
Together with DNA typing of the FY*A-, FY*B-, and
FY*Fy-associated polymorphisms by previously published PCR-
RFLP methods (Fig 5B), these results
indicated that the C286T substitution is associated with the
Fybweak phenotypes [Fy(abweak) and
Fy(a+bweak)] and confirmed the FY*X/*X genotype of
donor BAR and the FY*Fy/*X genotype of donors SEV, BE.T, and
TAR.
View larger version (31K):
[in this window]
[in a new window]
| Fig 5.
Effect of DARC Arg89Cys substitution on the binding of
anti-Fy antibodies and chemokine to COS-7 cell transfectants.
COS-7 cells were transiently transfected by the pcDNA3 expression
vector alone ( ) and the recombinant vectors containing the cDNA
encoding the Fyb (Arg89) ( ), or
Fyx (Cys89) ( ) DARC protein. Transfectant
cells were analyzed for Duffy antigen expression by flow cytometry with
anti-Fyb PoAb and anti-Fy3 and anti-Fy6 MoAbs and for
chemokine binding with 125I-IL-8, as described in Materials
and Methods. Specific binding of 125I-IL-8 yielded 100,000, 45,000, and 2,500 cpm with Fyb, Fyx, and mock
transfectants, respectively.
|
|
Furthermore, complete FY genotyping of BE.T family showed the
inheritance of the C286T substitution with that of weak Fyb
expression and demonstrated that the apparent paternity exclusion, suggested by the expression of Fya on RBCs from both
parents but not from the propositus, resulted from the presence of the
silent FY*B allele, FY*Fy., in the father (BE.L).
Binding of anti-Fy antibodies and chemokine to transfected COS-7
cells.
The C286T substitution identified in the FY*X allele, as
defined above, was introduced by site-directed mutagenesis in an expression vector carrying the Fyb cDNA,32 and
simian COS-7 cells were transiently transfected by the two recombinant
plasmids, pcDNA-Fyx and pcDNA-Fyb. Several
independent experiments were performed, and transfectants were further
analyzed only when transfection efficiency by the two constructs were
similar (4 experiments). Flow cytometry analysis showed that the
Fyx transfectants stained poorly with the anti-Fy3 (75%
reduction) and anti- Fy6 (70% reduction) MoAbs compared with the
Fyb transfectants (Fig 5). Reactivity of
anti-Fyb was also significantly lower on Fyx
transfectants, but this polyclonal reagent showed variable reduction of
Fyb expression between the different experiments, ranging
from 35% to 60%. Calculation of the anti-Fy3 and anti-Fy6 apparent
binding site numbers suggested that about 200,000 and 60,000 copies of DARC were expressed at the surface of Fyb and
Fyx transfectants, respectively. Binding of
125I-labeled IL-8 to transfected cells, performed in
parallel to Fy antigen expression analysis, showed a 55% reduction in
the chemokine binding capacity of Fyx transfectant compared
with Fyb transfectants (Fig 4).
| |
DISCUSSION |
The Fyx antigen.
Whereas the previous analyses of the Fyx phenotype were
based on barely quantitative agglutination studies, we have performed flow cytometry and chemokine binding analysis to accurately estimate the variation of Fy antigen expression on RBCs from different phenotypes. Using HD50 agglutination assay and human polyclonal antisera, Habibi et al27 previously found a full
correlation between Fyb and Fy3 depression on
Fyx RBCs. We confirmed these results with the four
Fy(abweak) samples, which indicated that these cells
exhibited a typical Fyx phenotype, as previously
defined.23,27 Fy5 expression could not be analyzed because
anti-Fy5 was not available. However, we demonstrated that there is also
a full correlation between Fy3 and Fy6 expression on RBCs of different
phenotypes, including Fyx cells (R = .98, data not
shown). Furthermore, by the use of murine MoAbs that allow antigen site
density to be precisely estimated, we calculated that, as compared with
FY*B/*B control cells, FY*X/*X RBCs expressed only 12%
of Fy3 and Fy6 antigens. The decrease in Fyb expression, as
measured with human anti-Fy b antisera, was slightly
higher, but the polyclonal nature of the reagent did not allow an
accurate calculation of Fyb site numbers.
Apart from its biological relevance (see below), chemokine binding to
RBCs also represents a powerful approach in the comparison of DARC
expression on RBCs with different phenotypes. It has been previously
shown that chemokines from both C-C and C-X-C classes bind to RBCs from
the three common Fy-positive phenotypes, but not to Fy-negative
RBCs.7 We show here that these chemokines bind also to
Fyx RBCs, but that the FY*X/*X RBCs could only bind
20% to 30% of the amount of chemokines that bind to FY*B/*B
RBCs. It is noteworthy that both flow cytometry and chemokine binding
analysis allowed us to discriminate between FY*X/*X and
FY*Fy/*X RBCs and that cells mistyped as Fy-negative could be
clearly reclassified as Fy-positive.
Recent structure-function analysis of DARC showed that the binding of
the various ligands used in this study involved different domains of
DARC. The Fy6 epitope has been precisely mapped by synthetic
peptide/pins technology and mutagenesis analysis to the heptapeptide
comprising residues Q19-25 of the NH-2 terminal extracellular
domain.32,35 The epitopes recognized by
anti-Fyb and anti-Fy3 have not been fully characterized.
However, the Fyb epitope is thought to encompasse the
polymorphic G42D position in the NH-2 terminal region associated with
the Fya/Fyb antigenic
polymorphism9,18-20 and analysis of chimeric receptors indicated that the third extracellular loop of DARC is necessary for
the binding of anti-Fy3.36 We have recently demonstrated that the Fy6, Fya/b, and Fy3 epitopes are all involved in
the binding of chemokines and that the close association of the first
and fourth extracellular domains of DARC by a disulfide bond is
required for ligand binding, because it may create a chemokine binding
pocket.32 Thus, the ability of Fyx RBCs to bind
all the anti-Fy antibodies and chemokines tested indicated that the
overall structure of the DARC polypeptide expressed at the membrane of
these cells is most likely not altered. Conversely, the parallel
decrease of the binding capacity for all ligands together with the
highly reduced amount of DARC polypeptide detected in Fyx
RBC membrane by Western blot analysis strongly suggested that the
Fyx antigen represents a poorly expressed but grossly
normal Fyb antigen.
The FY*X allele.
Not only has the structure of the FY*X allele not been
previously characterized, but whether "Fyb weak
reactions lies with Fyx being the tail-end expression of
Fyb37 or with it being the expression of a fourth
allele,"23 also remained an unresolved matter of
controversy. Sequence analysis of the FY transcripts showed that the 4 Fy(abweak) donors investigated in this study carry the
same allele, the coding sequence of which differs from that of a normal
FY*B allele by a single substitution, C286T. Further sequence
analysis of the FY gene did not show mutation in the intronic
sequence or in the promoter region that could account for
transcriptional or posttranscriptional alteration of FY
expression. Conversely, the C286T mutation is most likely causative of
low expression of DARC in Fy(abweak) erythroid cells,
because expression of the FY gene linked to an heterologous
promoter demonstrated that the construct with the Fybweak
sequence was expressed about twofold to threefold less in COS-7 cells
compared with the Fyb construct.
These results provide the definitive proof that the Fybweak
phenotype is conveyed by a fourth allele at the FY locus, FY*X
(GenBank accession no. AF055992), which is as distinct from the
FY*B allele as is the FY*Fy allele in Africans and
Afro-Americans. However, the present characterization of FY*X,
together with that of FY*A, FY*B,9,18-20
and FY*Fy in blacks21,22 and
Caucasians20 does not complete the elucidation of the
molecular genetic basis the Duffy blood group system. Indeed, the lack
of mutation in the sequence encoding for the minor, unspliced, isoform
of DARC in 2 unrelated Fy(abweak) donors investigated by
Mallinson et al20 suggested that the Fybweak
phenotype might arise from at least two different genetic mechanisms. However, the sequences of the promoter, exon 1, and intron 1 of FY was not elucidated when these investigators performed their study. Hence, it remains to be determined whether mutation in the 5
part of FY accounts for quantitative or qualitative alteration of Fyb expression in the 2 Fybweak donors
studied. A further complexity of the Duffy blood group system was
recently suggested by Shimizu et al38 in the course of a
serotyping study of 434 individuals from several Thai ethnic groups.
The investigators emphasized the presence in 8 individuals of a
weak-Fya antigen that has never been described before. It
is anticipated that these studies could lead to the characterization of
a fifth allele at the FY locus, FY*Aweak, which
should be to FY*A what FY*X is to FY*B.
FY*X DNA typing in clinical and transfusion medicine.
A FY*X DNA typing based on the C286T substitution has been
developed that, together with the previously published FY*A,
FY*B, and FY*Fy genotyping, can discriminate between
alleles associated with normal (FY*B), bone marrow silent
(FY*Fy), and weak (FY*X) expression of the
Fyb antigen and made a full FY genotyping of a large series
of donors easy to perform. Because FY*B/*B and FY*B/*X
RBCs are generally indistinguishable and because Fyx is
often undetetected in FY*A/*X and FY*Fy/*X RBCs by
standard serological methods, the frequency of FY*X will
certainly appear to be much higher than previously reported
(0.015)39 when population studies are performed using DNA
typing. Interestingly, by using the FY*B genotyping test,
Murphy et al40 have already reported that weakened
expression of the Fyb antigen is responsible for 12% of
discrepancies between the genotypically (FY*A/*B) and
serologically [Fy(a+b)] determined Fyb status of 109 Caucasian donors. It is assumed that complete FY genotyping
could show the presence of FY*X in most of these discrepancies.
In the present study, FY genotyping was used to demonstrate
that the inheritance of the silent FY*Fy allele accounts for
apparent paternity exclusion in family BE. Similarly, FY*X DNA
typing will be useful to investigate apparent anomalous inheritance
within the Duffy system in Caucasian families, which in most cases is due to weakened expression of Fyb.23
Anti-Fya and, to a lesser extent, anti-Fyb
antibodies can cause HDN, with some of them being fatal,1
and a recent study has pointed out the clinical value of antenal
Fya/Fyb genotyping in pregnancies at risk of
HDN due to anti-Fya.41 It is expected that the
Fyx genotyping test will also prove to be useful in the
management of pregnancies at risk of Fy hemolytic disease by
discriminating between fetuses with normal or weakened expression of
the Fyb antigen when the mother exhibits high titer of
anti-Fyb.
Even though no transfusion reaction related symptoms have yet been
observed when blood units with weakened expression of Fyb
were transfused to Fyb-negative patients, FY DNA typing
would have important implications with respect to the good practice of
blood transfusion.40 In that respect, the newly described
Fyx DNA test should be performed to ascertain that
Fyb-negative patients, with a high titer of
anti-Fyb, will be transfused by true
Fyb-negative blood units and not by Fybweak
samples serologically mistyped as Fyb-negative. Secondly,
it is important that RBCs serving as reference for blood bankers are
better characterized, both at the phenotypic and genetic levels.
Genotype-phenotype relationship.
The C286T substitution results in an Arg89Cys amino acid change that is
predicted to occur in the first cytoplasmic loop of the DARC
polypeptide, according to the current seven transmembrane domain
topological model.7 Thus, the elucidation of the
Fyx-associated substitution represents the first study that
highlights the critical role of intracellularly exposed residues in the
expression of DARC. The cytoplasmic localization of the
Fyx-specific amino acid (Cys89) fits well with the fact
that antibodies specific to Fyx RBCs have never been
characterized.
Because our data suggest that the overall structure of the
membrane-expressed Fyx polypeptide is similar to that of
the other allelic forms of DARC (see above), we assume that the
presence of a Cysteine residue at position 89 of the Fyx
polypeptide is not associated with an additional disulfide bond that
would significantly modify the tertiary structure of DARC.
In agreement with the positive inside rule,42 the three
short intracellular loops connecting the seven hydrophobic
transmembrane helix of DARC contain several positively charged residues
(Arg, Lys) that might be in the vicinity of the polar groups of the phospholipid molecules along the cytoplasmic side of the lipid bilayer.
It has been experimentally demonstrated that basic amino acids, through
their interaction with negatively charged phospholipids, are critical
in the blocking of the translocation of loops across the membrane and
thereby should contribute to the control of membrane topology (van
Klompenburg et al43 and references herein).
Accordingly, we suggest that, by modifying the positive charge of the
first intracellular loop, the lack of Arg89, a residue conserved (or replaced by the homologous charged residue Histidine) in the DARC related polypeptides from 11 nonhuman primate species, mouse, and
cow18,44 (and our unpublished results), might
result in an inefficient insertion of the Fyx polypeptide
in the cell membrane and thus account for the very low cell surface
expression of DARC in Fyx cells.
The Fyx phenotype and the elusive DARC
function.
The precise function of DARC in immunobiology and neurobiology remains
uncertain. Because a murine DARC-like protein with conserved chemokine
binding properties has been recently characterized,44 it is
expected that DARC-deficient mice will be obtained and will help in the
understanding of the biological role of DARC. Another approach to
determine how much DARC is important in normal and pathological human
physiology is to look for a potential correlation between the clinical
status and the Duffy phenotypes of a large serie of donors. Hence, the
fact that a large majority of blacks do not express DARC on their RBCs
without apparent clinical consequence strongly suggested that DARC on
RBCs is dispensable.6,45 On the other hand, the
demonstration that, in these Fy(ab) individuals, the
downregulation of DARC was restricted to erythroid
cells6,11,21 and thus should represent an adaptative
response to resist malaria, reinforced the hypothesis of an important
role of DARC in nonerythroid tissues. Conversely, this suggestion
became more difficult to support when it was demonstrated that at least
3 apparently healthy Fy(ab) Caucasians carry genomic mutations
that should results in the lack of DARC in all cells and
tissues.20 However, significant, albeit reduced expression
of Fya and Fy6 antigens (~40% as compared with control)
were achieved when one of these defective genes that carry a 14-bp
deletion resulting in a premature stop signal at codon 118 was
transfected in COS-7 cells, but, as expected from structure
function/analysis (see above), Fy3 was not expressed and chemokine did
not bind to this protein lacking extracellular loops 2 and 3 (our
unpublished results). Taken together, these results
indicated that, even though a truncated DARC-related protein might be
expressed in nonerythroid cells of these rare Caucasian Fy(ab)
donors, these donors failed to express a functional chemokine receptor
in all their tissues without detectable adverse consequences.
Importantly, one finding from the present study, as substantiated by
transfectant analysis, is that the substitution identified in the
FY*X allele is also predicted to alter DARC expression in all
tissues without deleterious consequences for normal physiology. If DARC
is involved in transduction of a signal across the membrane upon
chemokine binding, by a still undetermined pathway, it is not unlikely
that its residual level in homozygous Fyx cells could be
sufficient to support normal function. However, it has thus been
demonstrated that mutants of the IL-8 RA receptor that poorly bind
IL-8 could mediate signal transduction in a way similar
to how wild-type receptor does.46
In conclusion, the elucidation of the molecular basis of the
Fyx phenotype adds one more degree of complexity to the
genetics of the Duffy blood group system, but again raises questions
about the importance of DARC in erythroid and nonerythroid tissues. Indeed, if one tries to link up the genetics with biology, it must be
postulated that, whatever the precise function of DARC, other
structures might operate when it is poorly expressed or absent. Similar
compensatory mechanisms have been postulated to account for the
observation that inactivation of the water channel AQP-1 gene
in individuals with the Colton nul [Co(ab)] blood group
phenotype is not associated with any apparent clinical
consequence.47 Obviously, the challenge now is to develop
direct experimental approaches to elucidate the biological role of
DARC.
| |
FOOTNOTES |
Submitted March 19, 1998;
accepted May 18, 1998.
Address reprint requests to Yves Colin, PhD, INSERM U76, Institut
National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France; e-mail: ycolin{at}infobiogen.fr.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are indebted to Dr F. Buffiere, Dr M. Uchikawa, and Dr D. Blanchard for supplying the anti-Fya, anti-Fy3, and
anti-Fy6 MoAbs, respectively. We thank Dr P. Bailly for providing the
anti-P55 polyclonal antibody and Dr A. Proudfoot for the gift of
recombinant IL-8.
| |
REFERENCES |
1. Mollison PL, Engelfriet CP, Contreras M: Blood Transfusion in
Clinical Medicine (ed 10). Oxford, UK, Blackwell, 1997
2.
Miller LH,
Mason SJ,
Dvorak JA,
Mc Ginniss MH,
Rothman IK:
Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants.
Science
189:561,
1975[Abstract/Free Full Text]
3.
Miller LH,
Mason SJ,
Clyde DF,
Mc Ginniss MH:
The resistance factor to plasmodium vivax in blacks. The Duffy blood group genotype, FyFy.
N Engl J Med
295:302,
1975[Abstract]
4.
Darbonne WC,
Rice GC,
Mohler MA,
Apple A,
Hébert CA,
Valente AJ,
Baker JB:
Red blood cells as sink for interleukin-8, a leukocyte chemotaxin.
J Clin Invest
88:1362,
1991
5.
Horuk R,
Chitnis CE,
Darbonne WC,
Colby TJ,
Rybicki A,
Hadley TJ,
Miller LH:
A receptor for the malarial parasite Plasmodium vivax: The erythrocyte chemokine receptor.
Science
261:1182,
1993[Abstract/Free Full Text]
6.
Chaudhuri A,
Polyakova J,
Zbrzezna A,
Williams K,
Gulati S,
Pogo AO:
Cloning of glycoprotein D cDNA which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite.
Proc Natl Acad Sci USA
90:10793,
1993[Abstract/Free Full Text]
7.
Neote K,
Mak JY,
Kolakowski LF Jr,
Schall TJ:
Functional and biochemical analysis of the cloned Duffy antigen: Identity with the red blood cell chemokine receptor.
Blood
84:44,
1994[Abstract/Free Full Text]
8.
Chaudhuri A,
Zbrzezna V,
Polyakova J,
Pogo AO,
Hesselgessr J,
Horuk R:
Expression of the Duffy antigen in K562 cells.
J Biol Chem
269:10793,
1994
9.
Tournamille C,
Le Van Kim C,
Gane P,
Cartron JP,
Colin Y:
Molecular basis and PCR-DNA typing of the Fya/Fyb blood group polymorphism.
Hum Genet
95:407,
1995[Medline]
[Order article via Infotrieve]
10.
Hadley TJ,
Lu Z,
Wasniowska K,
Martin AW,
Peiper SC,
Hesseigesser J,
Horuk R:
Postcapillary venule endothelial cells in kidney express a multispecific chemokine receptor that is structurally and fonctionally identical to the erythroid isoform, which is the Duffy blood group antigen.
J Clin Invest
88:985,
1994
11.
Peiper SC,
Wang Z,
Neote K,
Martin AW,
Showel HJ,
Conklyn MJ,
Ogborne K,
Hadley TJ,
Lu ZH,
Hesselgesser J,
Horuk R:
The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor.
J Exp Med
81:1311,
1995
12.
Chaudhuri A,
Soren N,
Elkjaer ML,
Zbrzezna V,
Fang F,
Pogo OA:
Detection of Duffy antigen in the plasma membranes and caveolae of vascular endothelial and epithelial cells of nonerythroid organs.
Blood
89:701,
1997[Abstract/Free Full Text]
13.
Horuk R,
Martin A,
Hesselgesser J,
Hadley TJ,
Lu ZH,
Zi-xuan W,
Peiper SC:
The Duffy antigen receptor for chemokines: Structural analysis and expression in the brain.
J Leukoc Biol
59:29,
1996[Abstract]
14.
Le Van Kim C,
Tournamille C,
Kroviarski Y,
Cartron JP,
ColinY:
The 1.35 kb and 7.5 kb Duffy mRNA isoforms are differently regulated in various regions of brain, differ by the length of their 5 untranslated sequence but encode the same polypeptide.
Blood
90:2851,
1997[Free Full Text]
15.
de Winter RJ,
Manten A,
de Jong YP,
Adams R,
van Deventer SJH,
Lie KI:
Interleukin 8 released after acute myocardial infarction is mainly bound to erythrocytes.
Heart
78:598,
1997[Abstract/Free Full Text]
16.
Middleton J,
Neil S,
Wintle J,
Clark-Lewis I,
Moore H,
Lam C,
Auer M,
Hub E,
Rot A:
Transcytosis and surface presentation of IL-8 by venular endothelial cells.
Cell
91:385,
1997[Medline]
[Order article via Infotrieve]
17.
Iwamoto S,
Li J,
Omi T,
Ikemoto S,
Kajii E:
Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary endothelium.
Blood
87:378,
1996[Abstract/Free Full Text]
18.
Chaudhuri A,
Polyakova J,
Zbrzezna V,
Pogo OA:
The coding sequence of Duffy blood group gene in human and simians: Restriction fragment length polymorphism, antibody and malaria specificities, and expression in non erythroid tissues in Duffy-negative individuals.
Blood
85:615,
1995[Abstract/Free Full Text]
19.
Iwamoto S,
Omi T,
Kajii E,
Ikemoto S:
Genomic organization of the glycoprotein D gene: Duffy blood group Fya/Fyb alloantigen system is associated with a polymorphism at the 44-amino acid residue.
Blood
85:622,
1995[Abstract/Free Full Text]
20.
Mallinson G,
Soo KS,
Schall TJ,
Pisacka M,
Anstee DJ:
Mutations in the erythrocyte chemokine receptor (Duffy) gene: The molecular basis of the Fya/Fyb antigens and identification of a deletion in the Duffy gene of an apparently healthy individual with the Fy(ab) phenotype.
Br J Haematol
90:823,
1995[Medline]
[Order article via Infotrieve]
21.
Tournamille C,
Colin Y,
Cartron JP,
Le Van Kim C:
Disruption of a GATA motif in the Duffy gene promotor abolishes erythroid gene expression in Duffy-negative individuals.
Nat Genet
10:224,
1995[Medline]
[Order article via Infotrieve]
22.
Iwamoto S,
Li J,
Sugimoto N,
Okuda H,
Kajii E:
Characterization of the Duffy gene promotor: Evidence for tissue-specific abolishment of expression in Fy(ab) of black individuals.
Biochem Biophys Res Commun
222:852,
1996b[Medline]
[Order article via Infotrieve]
23.
Chown B,
Lewis M,
Kaita H:
The Duffy blood group system in Caucasians: Evidence for a new allele.
Am J Hum Genet
17:384,
1965[Medline]
[Order article via Infotrieve]
24.
Cedergren B,
Giles MC:
An Fyx Fyx individual found in northern Sweden.
Vox Sang
24:264,
1973
25. Marsh WL: Present status of the Duffy blood group system.
CRC Rev Clin Lab Sci 5:387, 1975
26.
Buchanan DI,
Sinclair M,
Sanger R,
Gavin J,
Teesdale P:
An Alberta Cree Indian with a rare Duffy antibody, anti-Fy3.
Vox Sang
30:114,
1976[Medline]
[Order article via Infotrieve]
27.
Habibi B,
Perrier P,
Salmon C:
HD50 assay evaluation of the antigen Fy 3 depression in Fyx individuals.
J Immunogenet
7:191,
1980[Medline]
[Order article via Infotrieve]
28.
Nichols ME,
Rubinstein P,
Barnwell J,
Rodriguez de Cordoba S,
Rosenfield R:
A new human blood group specificity define by a murine monoclonal antibody. Immunogenetics and association with susceptibility to Plasmodium vivax.
J Exp Med
166:776,
1987[Abstract/Free Full Text]
29.
Alouani S,
Gaertner HF,
Mermod JJ,
Power CA,
Bacon KB,
Wells TNC,
Proudfoot AEI:
A fluorescent interleukin-8 receptor probe produced by targetted labelling at the amino terminus.
Eur J Biochem
227:328,
1995[Medline]
[Order article via Infotrieve]
30.
Ruff P,
Speicher DW,
Husain-Chisti A:
Molecular identification of a major palmitoylated erythrocyte membrane protein containing the src homology 3 motif.
Proc Natl Acad Sci USA
88:6595,
1991[Abstract/Free Full Text]
31.
Lozano EM,
Grace O,
Romanowski V:
Isolation of RNA from whole blood for reliable use in RT-PCR amplification.
Trends Genet
9:296,
1993[Medline]
[Order article via Infotrieve]
32.
Tournamille C,
Le Van Kim C,
Gane P,
Blanchard D,
Proudfoot AEI,
Cartron JP,
Colin Y:
Close association of the first and fourth extracellular domains of the duffy antigen/receptor for chemokines by a disulfide bond is required for ligand binding.
J Biol Chem
272:16274,
1997[Abstract/Free Full Text]
33.
Steck TL,
Kant JA:
Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes.
Methods Enzymol
31:172,
1974[Medline]
[Order article via Infotrieve]
34.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680,
1970[Medline]
[Order article via Infotrieve]
35.
Wasniowska K,
Blanchard D,
Janvier D,
Wang Z,
Peiper SC,
Hadley TJ,
Lisowska E:
Identification of the Fy6 epitope recognized by two monoclonal antibodies in the N-terminel extracellular portion of the Duffy antigen receptor for chemokines.
Mol Immunol
33:917,
1996[Medline]
[Order article via Infotrieve]
36.
Lu ZH,
Zi-xuan W,
Horuk R,
Hesselgesser J,
Yan-chun L,
Hadley TJ,
Peiper SC:
The promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines is primarily localized to sequences in the amino- terminal domain.
J Biol Chem
270:26239,
1995[Abstract/Free Full Text]
37. Habibi B, Fouillade MT, Levanra D, Federspiel M, Salmon C:
Antigene Fyx: Étude quantitative chez les sujets
Fyb Fyx, Fya Fyx et
Fyx Fyx provenant de deux nouvelles familles.
Rev Fr Transfus Immuno-hématol 3:427, 1977
38.
Shimizu Y,
Kimura M,
Settheetham-Ishida W,
Duangchang P,
Ishida T:
Serotyping of Duffy blood group in several Thai ethnic groups.
Southeast Asian J Trop Med Public Health
28:32,
1997[Medline]
[Order article via Infotrieve]
39.
Lewis M,
Kaita H,
Chown B:
The Duffy blood group system in caucasians.
Vox Sang
23:523,
1972[Medline]
[Order article via Infotrieve]
40.
Murphy MT,
Templeton LJ,
Fleming J,
Fergusson M,
Peterkin M,
Fraser RH:
Comparison of Fyb status as determined serologically and genetically.
Transfus Med
7:135,
1997[Medline]
[Order article via Infotrieve]
41.
Goodrick MJ,
Hadley AG,
Poole G:
Haemolytic disease of the fetus and newborn due to anti-Fy a and the potential clinical value of Duffy genotyping in pregancies at risk.
Transfus Med
7:301,
1997[Medline]
[Order article via Infotrieve]
42.
Gavel Y,
Steppuhn J,
Herrmann R,
von Heijne G:
The "positive-inside rule" applies to thylakoid membranes proteins.
FEBS Lett
282:41,
1991[Medline]
[Order article via Infotrieve]
43.
van Klompenburg W,
Nilsson I,
von Heijne G,
de Kruijff B:
Anionic phospholipids are determinants of membrane protein topology.
EMBO J
16:4261,
1997[Medline]
[Order article via Infotrieve]
44.
Luo H,
Chaudhuri A,
Johnson KR,
Neote K,
Zbrzezna V,
He Y,
Pogo OA:
Cloning, characterisation, and mapping of a murine promiscuous chemokine receptor gene: Homolog of the human Duffy gene.
Genome Res
7:932,
1997[Abstract/Free Full Text]
45.
Chaudhuri A,
Zbrzezna V,
Johnson C,
Nichols ME,
Rubinstein P,
Marsh WL,
Pogo AO:
Purification and characterization of an erythrocyte membrane protein complex carrying Duffy blood group antigen.
J Biol Chem
264:13770,
1989[Abstract/Free Full Text]
46.
Leong SR,
Kabakoff RC,
Hébert CA:
Complete mutagenesis of the extracellular domain of interleukin-8 (IL-8) type A receptor identifies charged residues mediating IL-8 binding and signal transduction.
J Biol Chem
269:19343,
1994[Abstract/Free Full Text]
47.
Preston GM,
Smith BL,
Zeidel ML,
Moulds JJ,
Agre P:
Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.
Science
265:1585,
1994[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
J. S. Lee, C. W. Frevert, D. R. Thorning, S. Segerer, C. E. Alpers, J.-P. Cartron, Y. Colin, V. A. Wong, T. R. Martin, and R. B. Goodman
Enhanced Expression of Duffy Antigen in the Lungs During Suppurative Pneumonia
J. Histochem. Cytochem.,
February 1, 2003;
51(2):
159 - 166.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Tamasauskas, V. Powell, K. Saksela, and K. Yazdanbakhsh
A homologous naturally occurring mutation in Duffy and CCR5 leading to reduced receptor expression
Blood,
June 1, 2001;
97(11):
3651 - 3654.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Sidoux-Walter, N. Lucien, R. Nissinen, P. Sistonen, S. Henry, J. Moulds, J.-P. Cartron, and P. Bailly
Molecular heterogeneity of the Jknull phenotype: expression analysis of the Jk(S291P) mutation found in Finns
Blood,
August 15, 2000;
96(4):
1566 - 1573.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. A. Zimmerman, I. Woolley, G. L. Masinde, S. M. Miller, D. T. McNamara, F. Hazlett, C. S. Mgone, M. P. Alpers, B. Genton, B. A. Boatin, et al.
Emergence of FY*Anull in a Plasmodium vivax-endemic region of Papua New Guinea
PNAS,
November 23, 1999;
96(24):
13973 - 13977.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Yazdanbakhsh, S. Lee, Q. Yu, and M. E. Reid
Identification of a Defect in the Intracellular Trafficking of a Kell Blood Group Variant
Blood,
July 1, 1999;
94(1):
310 - 318.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract