Pierre Gane - Academia.edu (original) (raw)

Papers by Pierre Gane

Blood, 2002

In most cases, the lack of Rh in Rh(null) red cells is associated with RHAG gene mutations. We ex... more In most cases, the lack of Rh in Rh(null) red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter-based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-trans...

Transfusion, 2006

ABSTRACT Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing re... more ABSTRACT Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing reagents. Many blood group specificities, however, are not amenable to classic hybridoma technology. The phage display technology, aimed at isolating peptides or antibody fragments, offers an alternative strategy. Recombinant antibodies derived from this technology would greatly facilitate phenotyping and decrease analysis cost. A human single-chain Fv (scFv) phage-displayed library was panned on red blood cells (RBCs) in an attempt to isolate clones recognizing human RBC specificities. Three rounds of biopanning were performed. Enrichment was monitored by phage titration, and selected phage populations were analyzed further. Three major clones were identified by clone diversity analysis. One of them showed a specificity for Lua. This scFv was reconstructed into a human IgG1 by recombinant DNA methods. The reactivity of the reconstructed human IgG1 toward Lua is indistinguishable from its parent scFv. Moreover, the specificity of the antibody was confirmed by serologic assays, flow cytometry, and biochemical analysis with RBCs of different Lu phenotypes and a recombinant cell line expressing Lu glycoproteins. With phage display and standard recombinant DNA methods, isolation of a scFv of Lua specificity was successful, from which a complete human IgG1 MoAb of equivalent reactivity was reconstructed. To our knowledge, this is the first MoAb specific for Lua.

Transfusion Medicine Reviews, 1996

The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoprotein... more The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoproteins (Rh50, CD47, LW and glycophorin B); these interact in the red cell membrane and are lacking or severely reduced in Rhnull cells. As a result, individuals with Rhnull suffer chronic haemolytic anaemia known as the Rh-deficiency syndrome. Most frequently, Rhnull phenotypes are caused by homozygosity of an autosomal suppressor gene unlinked to the RH locus (Rhnull regulator or Rhmod types). We have analysed the genes and transcripts encoding Rh, CD47 and Rh50 proteins in five such unrelated Rhnull cases. In all patients, we identified alteration of Rh50--frameshift, nucleotide mutations, or failure of amplification--which correlated with Rhnull phenotype. We propose that mutant alleles of Rh50, which map to chromosome 6p11-21.1, are likely candidates for suppressors of the RH locus accounting for most cases of Rh-deficiency.

European Journal of Biochemistry, 2004

Abbreviations: a IIb b 3 CHO*, activated a IIb b 3 CHO cells; a IIb b 3 *, activated a IIb b 3 in... more Abbreviations: a IIb b 3 CHO*, activated a IIb b 3 CHO cells; a IIb b 3 *, activated a IIb b 3 integrin; LAD, leukocyte adhesion deficiency; L-cells, L929 mouse fibroblast cells; LW, Landsteiner-Wiener; mAb, monoclonal antibody; MAdCAM-1, mucosal addressin cell adhesion molecule 1; PMA, 4b-phorbol 12-myristate 13-acetate; RBC, red blood cell; VCAM-1, vascular cell adhesion molecule 1.

Molecular Membrane Biology, 1997

Endogenous phosphatidylserine (PS) exposure and lipid transport activity have been investigated f... more Endogenous phosphatidylserine (PS) exposure and lipid transport activity have been investigated for seven unrelated cases of Rhnull erythrocytes. Endogenous PS exposure was measured by prothrombinase activity. Out of six cases studied, two Rhnull samples exhibited abnormal aminophospholipid exposure, as suggested by the measurement of a lower Km of factor Xa for prothrombin. Aminophospholipid translocase activity was measured through the transbilayer redistribution of spin-labelled analogues of phospholipids. Provided that incubation conditions allow the maintainance of intracellular ATP level, no difference was observed between Rhnull and control erythrocytes, clearly indicating that the aminophospholipid translocase and Rh polypeptides are different molecular species.

Journal of Biological Chemistry, 2002

Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have ... more 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.

Journal of Biological Chemistry, 2002

AQP3 is a water and glycerol channel present on human erythrocytes and in various tissues. By pro... more AQP3 is a water and glycerol channel present on human erythrocytes and in various tissues. By protein and molecular biology analysis, two unrelated probands who developed alloantibodies to the high frequency antigen GIL were found to be AQP3-deficient. The defect is caused by homozygous mutation affecting the 5' donor splice site of intron 5 of the AQP3 gene. This mutation causes the skipping of exon 5 and generates a frameshift and premature stop codon. Functional studies by 90 degrees light scattering using a stopped-flow spectrometer revealed the absence of facilitated glycerol transport across red cell membranes from the probands, but the water and urea transports were normal. Expression studies into COS-7 cells followed by flow cytometry analysis showed that only cells transfected with AQP3 cDNA strongly reacted with anti-GIL antibodies. These findings represent the first reported cases of AQP3 deficiency in humans and provide the molecular basis of a new blood group system, GIL, encoded by the AQP3 protein.

European Journal of Immunogenetics, 2000

The McLeod syndrome is a rare X-linked recessive disorder characterized by blood group, neuromusc... more The McLeod syndrome is a rare X-linked recessive disorder characterized by blood group, neuromuscular and haematopoietic abnormalities. It is caused by XK gene defects and may include large deletions in the Xp21 region. Analysis of three unrelated McLeod patients for the presence of the XK, DMD, CYBB, ETX1, RPGR and OTC loci, as well as for the DXS709 marker, revealed deletions from the 39th exon of DMD to the ETX1 locus (patient Be), from the XK to RPGR loci (patient Bi) and from the XK to CYBB loci (patient Lh). All three patients normally expressed the Lutheran (Lu) red cell antigens, thus excluding the interval between the RPGR and DMD genes as site of the XS locus, previously mapped to the Xp21.2-Xq21.1 region and thought to regulate the expression of the LU blood group gene on chromosome 19.

Human Genetics, 1995

The Duffy blood group antigens are carried by the erythrocyte membrane glycoprotein gpD, which ha... more The Duffy blood group antigens are carried by the erythrocyte membrane glycoprotein gpD, which has a molecular weight of 35--45 kDa and which has been recently cloned. In this report, we have determined, at the nucleic acid level, the molecular basis for the blood group Fya/Fyb polymorphism. The gpD cDNAs isolated by reverse transcription/polymerase chain reaction (RT-PCR) from Fy(a+b-) and Fy(a-b+) donors differed by only one base susbstitution (G131A) changing Gly to Asp at position 44 of the gpD protein. When expressed in simian Cos-7 cells, the Fy(a+b--) and Fy(a-b+) gpD cDNA produce cell surface proteins that react with the anti-Fya and anti-Fyb antisera, respectively, demonstrating that they represent the FY*A and FY*B alleles of the Duffy blood group locus. The G131A nucleotide substitution has been correlated with a BanI restriction site polymorphism, which has allowed us to develop a method for the DNA typing of the main Duffy blood group antigens, by means of PCR/ restriction fragment length polymorphisms.

British Journal of Haematology, 2001

The linkage between blood group-related cell surface proteins and the detergent-insoluble materia... more The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIMassociated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

British Journal of Haematology, 1999

The time course expression of blood group antigens was examined by¯ow cytometry using a twophase ... more The time course expression of blood group antigens was examined by¯ow cytometry using a twophase liquid culture system that supports the proliferation and maturation of human erythroid progenitors from adult peripheral blood. The progression towards erythroid differentiation was followed by the expression changes of the transferrin receptor (CD71 ) and glycophorin A (GPA ). Four main categories of blood group markers were identi®ed: (i) those characterized by an early expression like ABO (A), Kell (K:2) and Rh50 which were detected in the Epoindependent phase 1, (ii) those including GPC (Gerbich, Ge antigens) and Fy6 which were expressed in the late phase 1, (iii) GPA (MN antigens), Wr b (Band 3/GPA interaction), Rh(D, Cc/Ee) and LW which appeared during the Epodependent phase 2 and (iv) those like Jk 3 and Lu b which were expressed in late phase 2.

British Journal of Haematology, 1996

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG ... more The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the r G r phenotype. Nucleotide sequence analysis revealed that the r G allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid r G gene (c-C-e or c-D-e) carries Ile60, Ser68 and Ser103 (as C and D polypeptides);

British Journal of Haematology, 2003

The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the... more The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fy a epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fy a mAbs to K562 cells respectively, Anti-Fy3 binding was abolished

British Journal of Haematology, 1998

We report a female patient whose Rh phenotype shifted from RhD-positive to RhD-negative over a 3-... more We report a female patient whose Rh phenotype shifted from RhD-positive to RhD-negative over a 3-year period (1991-94), during which time she was treated with mastectomy (1992) and local irradiation for a low-grade recurrent breast cancer. She was diagnosed with chronic myeloid leukaemia in 1994, and has since then received chemotherapy. The patient was repeatedly typed as O, RhD-positive between 1965 and 1991 and was repeatedly found RhD-negative after 1994. Bcr-Abl transcripts typical of Ph 1 chromosome were detected. Molecular analysis indicated that the patient was heterozygous at the RH locus, carrying one haplotype in which the RHD gene exhibited a single nucleotide deletion (G600) resulting in a frameshift and premature stop codon, and a normal RHCE gene (allele Ce). The second haplotype contained only the RHCE gene (allele ce) and was normal. Further analysis carried out on total leucocytes, purified neutrophils, EBVlymphoblastoid cell line and cultured erythroblasts indicated that the G600 deletion was restricted to the myeloid lineage. No modification of other blood group antigens could be detected. These findings suggest a somatic mutation which most probably occurred in a stem cell common to the myeloid lineage.

British Journal of Haematology, 1998

After testing red cells from 12 RhE variants with a panel of anti-E monoclonal antibodies (MoAbs)... more After testing red cells from 12 RhE variants with a panel of anti-E monoclonal antibodies (MoAbs), four patterns of reactivity were detected indicating that the MoAbs may recognize four distinct E epitopes designated epE1, epE2, epE3 and epE4. The variants were classified into four categories (cat EI to EIV) which carried epE1 and epE2, epE1 and epE4, epE1, epE3 and epE4, and all four epitopes, respectively. Molecular analysis of the transcripts and genomic DNA of the variants from cat EI, EII and EIII displayed three distinct genetic alterations. Cat EI variants exhibited a point mutation (T500A) in exon 4 of the RHCE gene that resulted in a Met167Lys substitution in the third extracellular loop of the RhcE protein. Cat EII variant carried a hybrid gene structure characterized by replacement of exons 1-3 (or 2-3) of the RHCE gene by their specific counterparts in the RHD gene. This latter variant was also associated with a weak expression of the RhC antigen. In cat EIII variants there was a partial DNA exchange of exon 5 sequences (nt 697 and 712) between the RHCE and the RHD genes, generating a hybrid Rh cE-D-cE protein carrying the Glu233 and Val238 substitutions. The serological and molecular studies of the RhE variants indicated that: (i) the RhE antigen is a mosaic composed of at least four epitopes and proline at position 226 is necessary but not sufficient for the full expression of the E antigen, (ii) the lack of RhE epitope(s) is associated with heterogenous molecular alterations of the RHCE gene, and (iii) amino-acids located on the third and fourth extracellular loops of the RhCE polypeptide are critical for some RhE epitopes expression.

Biochemical Journal, 2005

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammoni... more The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin-Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 microM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222-17227] constitute a family of NH3 channels in mammalian cells.

Blood, 2002

In most cases, the lack of Rh in Rh(null) red cells is associated with RHAG gene mutations. We ex... more In most cases, the lack of Rh in Rh(null) red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter-based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-trans...

Transfusion, 2006

ABSTRACT Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing re... more ABSTRACT Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing reagents. Many blood group specificities, however, are not amenable to classic hybridoma technology. The phage display technology, aimed at isolating peptides or antibody fragments, offers an alternative strategy. Recombinant antibodies derived from this technology would greatly facilitate phenotyping and decrease analysis cost. A human single-chain Fv (scFv) phage-displayed library was panned on red blood cells (RBCs) in an attempt to isolate clones recognizing human RBC specificities. Three rounds of biopanning were performed. Enrichment was monitored by phage titration, and selected phage populations were analyzed further. Three major clones were identified by clone diversity analysis. One of them showed a specificity for Lua. This scFv was reconstructed into a human IgG1 by recombinant DNA methods. The reactivity of the reconstructed human IgG1 toward Lua is indistinguishable from its parent scFv. Moreover, the specificity of the antibody was confirmed by serologic assays, flow cytometry, and biochemical analysis with RBCs of different Lu phenotypes and a recombinant cell line expressing Lu glycoproteins. With phage display and standard recombinant DNA methods, isolation of a scFv of Lua specificity was successful, from which a complete human IgG1 MoAb of equivalent reactivity was reconstructed. To our knowledge, this is the first MoAb specific for Lua.

Transfusion Medicine Reviews, 1996

The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoprotein... more The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoproteins (Rh50, CD47, LW and glycophorin B); these interact in the red cell membrane and are lacking or severely reduced in Rhnull cells. As a result, individuals with Rhnull suffer chronic haemolytic anaemia known as the Rh-deficiency syndrome. Most frequently, Rhnull phenotypes are caused by homozygosity of an autosomal suppressor gene unlinked to the RH locus (Rhnull regulator or Rhmod types). We have analysed the genes and transcripts encoding Rh, CD47 and Rh50 proteins in five such unrelated Rhnull cases. In all patients, we identified alteration of Rh50--frameshift, nucleotide mutations, or failure of amplification--which correlated with Rhnull phenotype. We propose that mutant alleles of Rh50, which map to chromosome 6p11-21.1, are likely candidates for suppressors of the RH locus accounting for most cases of Rh-deficiency.

European Journal of Biochemistry, 2004

Abbreviations: a IIb b 3 CHO*, activated a IIb b 3 CHO cells; a IIb b 3 *, activated a IIb b 3 in... more Abbreviations: a IIb b 3 CHO*, activated a IIb b 3 CHO cells; a IIb b 3 *, activated a IIb b 3 integrin; LAD, leukocyte adhesion deficiency; L-cells, L929 mouse fibroblast cells; LW, Landsteiner-Wiener; mAb, monoclonal antibody; MAdCAM-1, mucosal addressin cell adhesion molecule 1; PMA, 4b-phorbol 12-myristate 13-acetate; RBC, red blood cell; VCAM-1, vascular cell adhesion molecule 1.

Molecular Membrane Biology, 1997

Endogenous phosphatidylserine (PS) exposure and lipid transport activity have been investigated f... more Endogenous phosphatidylserine (PS) exposure and lipid transport activity have been investigated for seven unrelated cases of Rhnull erythrocytes. Endogenous PS exposure was measured by prothrombinase activity. Out of six cases studied, two Rhnull samples exhibited abnormal aminophospholipid exposure, as suggested by the measurement of a lower Km of factor Xa for prothrombin. Aminophospholipid translocase activity was measured through the transbilayer redistribution of spin-labelled analogues of phospholipids. Provided that incubation conditions allow the maintainance of intracellular ATP level, no difference was observed between Rhnull and control erythrocytes, clearly indicating that the aminophospholipid translocase and Rh polypeptides are different molecular species.

Journal of Biological Chemistry, 2002

Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have ... more 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.

Journal of Biological Chemistry, 2002

AQP3 is a water and glycerol channel present on human erythrocytes and in various tissues. By pro... more AQP3 is a water and glycerol channel present on human erythrocytes and in various tissues. By protein and molecular biology analysis, two unrelated probands who developed alloantibodies to the high frequency antigen GIL were found to be AQP3-deficient. The defect is caused by homozygous mutation affecting the 5' donor splice site of intron 5 of the AQP3 gene. This mutation causes the skipping of exon 5 and generates a frameshift and premature stop codon. Functional studies by 90 degrees light scattering using a stopped-flow spectrometer revealed the absence of facilitated glycerol transport across red cell membranes from the probands, but the water and urea transports were normal. Expression studies into COS-7 cells followed by flow cytometry analysis showed that only cells transfected with AQP3 cDNA strongly reacted with anti-GIL antibodies. These findings represent the first reported cases of AQP3 deficiency in humans and provide the molecular basis of a new blood group system, GIL, encoded by the AQP3 protein.

European Journal of Immunogenetics, 2000

The McLeod syndrome is a rare X-linked recessive disorder characterized by blood group, neuromusc... more The McLeod syndrome is a rare X-linked recessive disorder characterized by blood group, neuromuscular and haematopoietic abnormalities. It is caused by XK gene defects and may include large deletions in the Xp21 region. Analysis of three unrelated McLeod patients for the presence of the XK, DMD, CYBB, ETX1, RPGR and OTC loci, as well as for the DXS709 marker, revealed deletions from the 39th exon of DMD to the ETX1 locus (patient Be), from the XK to RPGR loci (patient Bi) and from the XK to CYBB loci (patient Lh). All three patients normally expressed the Lutheran (Lu) red cell antigens, thus excluding the interval between the RPGR and DMD genes as site of the XS locus, previously mapped to the Xp21.2-Xq21.1 region and thought to regulate the expression of the LU blood group gene on chromosome 19.

Human Genetics, 1995

The Duffy blood group antigens are carried by the erythrocyte membrane glycoprotein gpD, which ha... more The Duffy blood group antigens are carried by the erythrocyte membrane glycoprotein gpD, which has a molecular weight of 35--45 kDa and which has been recently cloned. In this report, we have determined, at the nucleic acid level, the molecular basis for the blood group Fya/Fyb polymorphism. The gpD cDNAs isolated by reverse transcription/polymerase chain reaction (RT-PCR) from Fy(a+b-) and Fy(a-b+) donors differed by only one base susbstitution (G131A) changing Gly to Asp at position 44 of the gpD protein. When expressed in simian Cos-7 cells, the Fy(a+b--) and Fy(a-b+) gpD cDNA produce cell surface proteins that react with the anti-Fya and anti-Fyb antisera, respectively, demonstrating that they represent the FY*A and FY*B alleles of the Duffy blood group locus. The G131A nucleotide substitution has been correlated with a BanI restriction site polymorphism, which has allowed us to develop a method for the DNA typing of the main Duffy blood group antigens, by means of PCR/ restriction fragment length polymorphisms.

British Journal of Haematology, 2001

The linkage between blood group-related cell surface proteins and the detergent-insoluble materia... more The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIMassociated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

British Journal of Haematology, 1999

The time course expression of blood group antigens was examined by¯ow cytometry using a twophase ... more The time course expression of blood group antigens was examined by¯ow cytometry using a twophase liquid culture system that supports the proliferation and maturation of human erythroid progenitors from adult peripheral blood. The progression towards erythroid differentiation was followed by the expression changes of the transferrin receptor (CD71 ) and glycophorin A (GPA ). Four main categories of blood group markers were identi®ed: (i) those characterized by an early expression like ABO (A), Kell (K:2) and Rh50 which were detected in the Epoindependent phase 1, (ii) those including GPC (Gerbich, Ge antigens) and Fy6 which were expressed in the late phase 1, (iii) GPA (MN antigens), Wr b (Band 3/GPA interaction), Rh(D, Cc/Ee) and LW which appeared during the Epodependent phase 2 and (iv) those like Jk 3 and Lu b which were expressed in late phase 2.

British Journal of Haematology, 1996

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG ... more The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the r G r phenotype. Nucleotide sequence analysis revealed that the r G allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid r G gene (c-C-e or c-D-e) carries Ile60, Ser68 and Ser103 (as C and D polypeptides);

British Journal of Haematology, 2003

The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the... more The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fy a epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fy a mAbs to K562 cells respectively, Anti-Fy3 binding was abolished

British Journal of Haematology, 1998

We report a female patient whose Rh phenotype shifted from RhD-positive to RhD-negative over a 3-... more We report a female patient whose Rh phenotype shifted from RhD-positive to RhD-negative over a 3-year period (1991-94), during which time she was treated with mastectomy (1992) and local irradiation for a low-grade recurrent breast cancer. She was diagnosed with chronic myeloid leukaemia in 1994, and has since then received chemotherapy. The patient was repeatedly typed as O, RhD-positive between 1965 and 1991 and was repeatedly found RhD-negative after 1994. Bcr-Abl transcripts typical of Ph 1 chromosome were detected. Molecular analysis indicated that the patient was heterozygous at the RH locus, carrying one haplotype in which the RHD gene exhibited a single nucleotide deletion (G600) resulting in a frameshift and premature stop codon, and a normal RHCE gene (allele Ce). The second haplotype contained only the RHCE gene (allele ce) and was normal. Further analysis carried out on total leucocytes, purified neutrophils, EBVlymphoblastoid cell line and cultured erythroblasts indicated that the G600 deletion was restricted to the myeloid lineage. No modification of other blood group antigens could be detected. These findings suggest a somatic mutation which most probably occurred in a stem cell common to the myeloid lineage.

British Journal of Haematology, 1998

After testing red cells from 12 RhE variants with a panel of anti-E monoclonal antibodies (MoAbs)... more After testing red cells from 12 RhE variants with a panel of anti-E monoclonal antibodies (MoAbs), four patterns of reactivity were detected indicating that the MoAbs may recognize four distinct E epitopes designated epE1, epE2, epE3 and epE4. The variants were classified into four categories (cat EI to EIV) which carried epE1 and epE2, epE1 and epE4, epE1, epE3 and epE4, and all four epitopes, respectively. Molecular analysis of the transcripts and genomic DNA of the variants from cat EI, EII and EIII displayed three distinct genetic alterations. Cat EI variants exhibited a point mutation (T500A) in exon 4 of the RHCE gene that resulted in a Met167Lys substitution in the third extracellular loop of the RhcE protein. Cat EII variant carried a hybrid gene structure characterized by replacement of exons 1-3 (or 2-3) of the RHCE gene by their specific counterparts in the RHD gene. This latter variant was also associated with a weak expression of the RhC antigen. In cat EIII variants there was a partial DNA exchange of exon 5 sequences (nt 697 and 712) between the RHCE and the RHD genes, generating a hybrid Rh cE-D-cE protein carrying the Glu233 and Val238 substitutions. The serological and molecular studies of the RhE variants indicated that: (i) the RhE antigen is a mosaic composed of at least four epitopes and proline at position 226 is necessary but not sufficient for the full expression of the E antigen, (ii) the lack of RhE epitope(s) is associated with heterogenous molecular alterations of the RHCE gene, and (iii) amino-acids located on the third and fourth extracellular loops of the RhCE polypeptide are critical for some RhE epitopes expression.

Biochemical Journal, 2005

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammoni... more The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin-Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 microM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222-17227] constitute a family of NH3 channels in mammalian cells.