Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia
1
202 Oral communications and posters / Transfusion Clinique et Biologique 17 (2010) 200–204 Methods.– Candidate genes were sequenced from DNA of the proband including STEAP3, an iron reductase involved in the iron-transferrin cycle and exocytosis. Quantitative RT PCR was performed from RNA. Results.– A premature STOP codon (c.C110X) was found in the STEAP3 gene at the heterozygous state in the proband. The same mutation was found in the two other affected siblings and their unaffected father. No abnormality was found in the maternal alleles. We studied the relative expression of STEAP3 alleles in immortalized lymphocytes from family members after treatment the cells with emetin to prevent the degradation of the mutated allele resulting from nonsense mediated mRNA decay. We found that the relative expression of the normal allele is about twice higher in the father than in his three affected offspring. Quantita- tive PCR from blood RNA confirmed these results and STEAP3 expression in blood from healthy unrelated volunteers was highly variable, allele specific and associated to 3’UTR SNPs. Conclusion.– STEAP3 deficiency leads to severe anaemia in human and may result from the genotypic combination of a mutated allele and a low expressed allele. Recent reports demonstrated that allele specific expression is a rather common finding that may concern 5-20% of the genes (including STEAP3). The coinheritance of a mutated and a low-expressed normal allele may mimic either a dominant or a recessive transmission depending on the frequency of the low expressed allele in the population. doi:10.1016/j.tracli.2010.06.017 CO-07 VHHs or nanobodies directed against proteins of the human red cell membrane D. Smolarek a , I. Habib a , C. Hattab b , S. Cochet a , G. Hassanzadeh-Ghassabeh c , C. Gutierrez d , J. Picot b , M. Grodecka e , K. Wasniowska e , R. Udomsangpetch f , A.G. de Brevern a , S. Muyldermans c , Y. Colin a , C. Le Van Kim a , M. Czerwinski e , O. Bertrand a,∗ a Inserm, Paris, France b INTS, Paris, France c Vrije Universiteit Brussel, Flanders interuniversity Institute of Biotechnology, Bruxelles, Belgium d Veterinary Faculty, Las Palmas, Spain e Polish Academy of Sciences, Wroclaw, Poland f Mahidol University, Bangkok, Thailand ∗ Corresponding author. E-mail: [email protected] (O. Bertrand). Aim.– VHHs, are recombinant derivatives of camelid immunoglobulins. Two IgG sub-classes of camelid immunoglobulins are composed of heavy chains- only and lack all light chains. Their variable regions are easily cloned from lymphocytes of the animals. VHHs, endowed with the properties of authentic antibodies are famed for a good expression yield and an excellent solubility. Methods.– To date, we produced VHHs after having immunized a dromedary against recombinant first extra cellular domain (ECD1) of Duffy Antigen Recep- tor for Chemokines (DARC) expressed in E. coli. Anti-glycophorin A VHHs were produced from lymphocytes from another dro- medary immunized by transfusion of human whole blood. Results.– Several anti DARC VHHs were obtained which recognize the Fy6 epitope. One of them studied in detail has a subnanomolar affinity for E. coli produced ECD1 and also for full size DARC purified from engineered eukaryotic cells. It does inhibit IL-8 binding to red cells, it does inhibit invasion of red cells by P. vivax merozoite. After immobilization, it can be used for one step purification of DARC from eukaryotic recombinant cells or human red cells. Produced anti-glycophorin A VHHs are presently not yet fully characterized, they might have several applications, e.g. as building blocks for new blood typing reagents or for erythrocytes autoagglutination assays. Conclusion.– Results obtained to date illustrate potentialities of VHHs as power- ful and innovative reagents for studies of red cells membrane proteins. doi:10.1016/j.tracli.2010.06.018 CO-08 Investigating band 3 multiprotein complex assembly during erythropoiesis T. Satchwell a,∗ , E. van den Akker a , G. Daniels b , A. Toye a a University of Bristol, Bristol, United Kingdom b National Health Service Blood and Transplant, Bristol, United Kingdom ∗ Corresponding author. E-mail: [email protected] (T. Satchwell). Aim.– Band 3 forms the core of a large multiprotein complex in the erythrocyte membrane, which also includes proteins of the Rhesus complex. We aim to investigate the assembly of this important macrocomplex during the process of erythropoiesis. Methods.– We used a synchronised human erythroid progenitor culture system to investigate the expression and localisation band 3 macrocomplex proteins during erythroid differentiation. Confocal imaging, flow cytometry and biochemical techniques were employed to investigate the sites of assembly and composition of these complexes in erythroid progenitors as they mature. Results.– We show that protein4.2 expression is evident early at the basophilic stage of erythropoiesis and occurs in tandem with band 3 expression. Proteolytic cleavage in conjunction with coimmunoprecipitations demonstrated that pro- tein 4.2 associates with band 3 initially in an intracellular compartment within differentiating erythroid progenitors. The association of Rh with RhAG is also observable at the plasma membrane at this stage whilst the intracellular pool of RhAG is depleted during differentiation as it is delivered to the surface. Unlike in erythrocytes no interaction between the Rh complex and band 3 was detectable. Conclusion.– We demonstrate that some of the key interactions within the band 3 macrocomplex occur early during erythropoiesis. Association of band 3 with the Rh complex may be weaker than in erythrocytes or occur at a later point, possibly during reticulocyte maturation. doi:10.1016/j.tracli.2010.06.019 CO-09 Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia S. Coulon a,∗ , P. Wang b , D. Grapton b , M. Dussiot a , C. Callens a , M. Benhamou b , M. Cogné c , O. Hermine a , I.C. Moura b a CNRS UMR 8147, Paris, France b Inserm U699, Paris, France c CNRS UMR 6101, Limoges, France ∗ Corresponding author. E-mail: [email protected] (S. Coulon). Aim.– Erythropoiesis is a tightly regulated process. Erythroblasts highly express the transferrin receptor 1 (TfR1), previously identified as an IgA1 receptor. Given that IgA1+ plasma cells are abundant in the bone marrow, we evaluated if IgA1 could play a role in the regulation of erythropoiesis. Methods.– We first evaluated the contribution of IgA1 to the proliferation and the clonogenicity of erythroid cells. We further explored the role of human pIgA1 in erythropoiesis in vivo. Results.– Here we show that TfR1-bound polymeric IgA1 (pIgA1) rescued the growth and clonogenic potential of erythroblasts under suboptimal Epo concentrations. The molecular mechanism involved magnification of Epo signa- ling through activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K/AKT) signaling pathways. In a humani- zed mouse model (1KI mice) IgA1 polymers accelerated stress erythropoiesis. By contrast, mice lacking the immunoglobulin joining-(J) chain (1KI/J-chain- /-) demonstrated a delayed recovery from anemia. pIgA1 levels increased upon hypoxia in mice and were high in patients under hypoxic stress. IgA1 oligome- rization status was modulated by TGF-ß1 produced by stromal cells subjected to hypoxia. Conclusion.– Therefore, hypoxia regulates pIgA1 production and, in turn, pIgA1/TfR1 signaling modulates erythroblast sensitivity to growth factors. Tar- geting this pathway could provide alternative approaches to treat anemia, in particular in Epo hypo-responsive patients. doi:10.1016/j.tracli.2010.06.020
Transcript of Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia
202 Oral communications and posters / Transfusion Clinique et Biologique 17 (2010) 200–204
Methods.– Candidate genes were sequenced from DNA of the proband includingSTEAP3, an iron reductase involved in the iron-transferrin cycle and exocytosis.Quantitative RT PCR was performed from RNA.Results.– A premature STOP codon (c.C110X) was found in the STEAP3 gene atthe heterozygous state in the proband. The same mutation was found in the twoother affected siblings and their unaffected father. No abnormality was foundin the maternal alleles. We studied the relative expression of STEAP3 alleles inimmortalized lymphocytes from family members after treatment the cells withemetin to prevent the degradation of the mutated allele resulting from nonsensemediated mRNA decay. We found that the relative expression of the normal alleleis about twice higher in the father than in his three affected offspring. Quantita-tive PCR from blood RNA confirmed these results and STEAP3 expression inblood from healthy unrelated volunteers was highly variable, allele specific andassociated to 3’UTR SNPs.Conclusion.– STEAP3 deficiency leads to severe anaemia in human and mayresult from the genotypic combination of a mutated allele and a low expressedallele. Recent reports demonstrated that allele specific expression is a rathercommon finding that may concern 5-20% of the genes (including STEAP3).The coinheritance of a mutated and a low-expressed normal allele may mimiceither a dominant or a recessive transmission depending on the frequency of thelow expressed allele in the population.
doi:10.1016/j.tracli.2010.06.017
CO-07
VHHs or nanobodies directed against proteins of thehuman red cell membraneD. Smolarek a, I. Habib a, C. Hattab b, S. Cochet a,G. Hassanzadeh-Ghassabeh c, C. Gutierrez d, J. Picot b, M. Grodecka e,K. Wasniowska e, R. Udomsangpetch f, A.G. de Brevern a, S. Muyldermans c,Y. Colin a, C. Le Van Kim a, M. Czerwinski e, O. Bertrand a,∗a Inserm, Paris, Franceb INTS, Paris, Francec Vrije Universiteit Brussel, Flanders interuniversity Institute ofBiotechnology, Bruxelles, Belgiumd Veterinary Faculty, Las Palmas, Spaine Polish Academy of Sciences, Wroclaw, Polandf Mahidol University, Bangkok, Thailand∗Corresponding author.E-mail: [email protected] (O. Bertrand).
Aim.– VHHs, are recombinant derivatives of camelid immunoglobulins. TwoIgG sub-classes of camelid immunoglobulins are composed of heavy chains-only and lack all light chains. Their variable regions are easily cloned fromlymphocytes of the animals. VHHs, endowed with the properties of authenticantibodies are famed for a good expression yield and an excellent solubility.Methods.– To date, we produced VHHs after having immunized a dromedaryagainst recombinant first extra cellular domain (ECD1) of Duffy Antigen Recep-tor for Chemokines (DARC) expressed in E. coli.Anti-glycophorin A VHHs were produced from lymphocytes from another dro-medary immunized by transfusion of human whole blood.Results.– Several anti DARC VHHs were obtained which recognize the Fy6epitope. One of them studied in detail has a subnanomolar affinity for E. coliproduced ECD1 and also for full size DARC purified from engineered eukaryoticcells. It does inhibit IL-8 binding to red cells, it does inhibit invasion of redcells by P. vivax merozoite. After immobilization, it can be used for one steppurification of DARC from eukaryotic recombinant cells or human red cells.Produced anti-glycophorin A VHHs are presently not yet fully characterized,they might have several applications, e.g. as building blocks for new blood typingreagents or for erythrocytes autoagglutination assays.Conclusion.– Results obtained to date illustrate potentialities of VHHs as power-ful and innovative reagents for studies of red cells membrane proteins.
doi:10.1016/j.tracli.2010.06.018
CO-08
Investigating band 3 multiprotein complex assemblyduring erythropoiesisT. Satchwell a,∗, E. van den Akker a, G. Daniels b, A. Toye a
a University of Bristol, Bristol, United Kingdomb National Health Service Blood and Transplant, Bristol, United Kingdom∗Corresponding author.E-mail: [email protected] (T. Satchwell).
Aim.– Band 3 forms the core of a large multiprotein complex in the erythrocytemembrane, which also includes proteins of the Rhesus complex. We aim toinvestigate the assembly of this important macrocomplex during the process oferythropoiesis.Methods.– We used a synchronised human erythroid progenitor culture system toinvestigate the expression and localisation band 3 macrocomplex proteins duringerythroid differentiation. Confocal imaging, flow cytometry and biochemicaltechniques were employed to investigate the sites of assembly and compositionof these complexes in erythroid progenitors as they mature.Results.– We show that protein 4.2 expression is evident early at the basophilicstage of erythropoiesis and occurs in tandem with band 3 expression. Proteolyticcleavage in conjunction with coimmunoprecipitations demonstrated that pro-tein 4.2 associates with band 3 initially in an intracellular compartment withindifferentiating erythroid progenitors. The association of Rh with RhAG is alsoobservable at the plasma membrane at this stage whilst the intracellular pool ofRhAG is depleted during differentiation as it is delivered to the surface. Unlike inerythrocytes no interaction between the Rh complex and band 3 was detectable.Conclusion.– We demonstrate that some of the key interactions within the band 3macrocomplex occur early during erythropoiesis. Association of band 3 with theRh complex may be weaker than in erythrocytes or occur at a later point, possiblyduring reticulocyte maturation.
doi:10.1016/j.tracli.2010.06.019
CO-09
Polymeric IgA1 controls erythroblast proliferation andaccelerates erythropoiesis recovery in anemiaS. Coulon a,∗, P. Wang b, D. Grapton b, M. Dussiot a, C. Callens a,M. Benhamou b, M. Cogné c, O. Hermine a, I.C. Moura b
a CNRS UMR 8147, Paris, Franceb Inserm U699, Paris, Francec CNRS UMR 6101, Limoges, France∗Corresponding author.E-mail: [email protected] (S. Coulon).
Aim.– Erythropoiesis is a tightly regulated process. Erythroblasts highly expressthe transferrin receptor 1 (TfR1), previously identified as an IgA1 receptor. Giventhat IgA1+ plasma cells are abundant in the bone marrow, we evaluated if IgA1could play a role in the regulation of erythropoiesis.Methods.– We first evaluated the contribution of IgA1 to the proliferation and theclonogenicity of erythroid cells. We further explored the role of human pIgA1in erythropoiesis in vivo.Results.– Here we show that TfR1-bound polymeric IgA1 (pIgA1) rescuedthe growth and clonogenic potential of erythroblasts under suboptimal Epoconcentrations. The molecular mechanism involved magnification of Epo signa-ling through activation of the mitogen-activated protein kinase (MAPK) andphosphatidylinositol-3-kinase (PI3K/AKT) signaling pathways. In a humani-zed mouse model (�1KI mice) IgA1 polymers accelerated stress erythropoiesis.By contrast, mice lacking the immunoglobulin joining-(J) chain (�1KI/J-chain-/-) demonstrated a delayed recovery from anemia. pIgA1 levels increased uponhypoxia in mice and were high in patients under hypoxic stress. IgA1 oligome-rization status was modulated by TGF-ß1 produced by stromal cells subjectedto hypoxia.Conclusion.– Therefore, hypoxia regulates pIgA1 production and, in turn,pIgA1/TfR1 signaling modulates erythroblast sensitivity to growth factors. Tar-geting this pathway could provide alternative approaches to treat anemia, inparticular in Epo hypo-responsive patients.
doi:10.1016/j.tracli.2010.06.020
