ALBERT

Description: Plasmodium falciparum–derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2–mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin–actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2–induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.

Description: Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 −/− red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 −/− red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.

Description: We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophageprotein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis. © 1998 by The American Society of Hematology.

Description: Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 −/− red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 −/− red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.

Description: We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophageprotein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis. © 1998 by The American Society of Hematology.

Description: In mammals, erythropoiesis takes place in distinct anatomic units called erythroblastic islands that are consisted of a central macrophage surrounded by erythroblasts of varying maturity. The attachment of erythroblasts to macrophages within these islands promotes terminal maturation and enucleation of erythroid cells. Among a few other adhesion molecules known to date, Emp (erythroblast macrophage protein) is involved in this association. Our recent gene targeting studies showed that in the absence of Emp, erythroblastic islands are not formed and erythroid cells do not undergo enucleation. In addition, Emp null macrophages do not fully mature. Information on the changes in the expression level and cellular localization of Emp in differentiating erythroid and macrophage cells is essential for understanding the function of Emp both in the formation of erythroblastic islands and in the development of these two cell types. Previously we showed that Emp is expressed in immature erythroid precursors but not in mature erythrocytes, consistent with the fact that mature red blood cells do not adhere. However, no information is available on the expression of Emp in macrophages. To address this issue, we have used primary mouse fetal liver macrophages cultured for various time periods. Our studies showed that while Emp was expressed in all stages of maturation, the localization pattern changed dramatically during maturation: in immature macrophages, a substantial fraction of Emp was intracellular, whereas in more mature cells, Emp was expressed largely at the plasma membrane. To determine if the membrane-associated Emp is exposed on the cell surface, we employed biotin labeling of surface proteins, and compared the fraction of total Emp that is accessible to the biotinylation reagent with the fraction that is inaccessible. Our data showed that approximately 5–20% of total cellular Emp was present on the surface of immature macrophage precursors compared to 60–70% in fully matured macrophages. Cell surface expression of Emp was further confirmed by live cell staining, without prior fixation, of macrophages at different stages of maturation. Intracellular pool of Emp was present largely in the nucleus where it co-localized with the nuclear matrix marker, the spliceosome assembly factor SC35. To examine the trafficking of newly synthesized Emp, we performed pulse-chase experiments in macrophages of varying maturity. We found that nascent Emp migrated intracellularly from the nucleus to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture showed that erythroid cells attached to mature macrophages but not to immature macrophage precursors. Taken together, our data shows that the temporal and spatial expression of Emp in erythroid and myeloid cells correlates with its involvement in the attachment of these two cell types in mammalian erythropoiesis. Furthermore, localization of Emp at multiple sites within the cellular environment suggests functions not limited to cell attachment.

Description: Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.

Description: Emp, erythroblast macrophage protein, was originally detected in erythroblasts and macrophages, which form erythroblastic islands during erythropoiesis in the human bone marrow. The physical contact between erythroblasts and macrophages was suggested to promote the terminal maturation of erythroblasts, leading to their enucleation in vitro. To evaluate the function of Emp in vivo, we employed gene targeting studies to develop an Emp(−/−) mouse model. Mouse embryonic stem cells containing a gene-trap insertion in Emp were obtained from BayGenomics. Insertion of the gene-trap vector into Emp was verified by direct sequencing of cDNA obtained by 5′RACE. Chimeric mice generated by blastocyst microinjection were intercrossed, and the offspring were genotyped by PCR and Southern hybridization. The Emp (+/−) mice were healthy and fertile. However, no live Emp (−/−) mice were found among the progeny of the Emp (+/−) intercrosses. Analysis of timed pregnancies revealed that Emp (−/−) embryos were present at a frequency roughly consistent with Mendelian inheritance throughout the embryonal stages. Homozygous Emp (−/−) embryos were small and pale compared to their littermates, and they survived embryonic development but died at birth. To determine the effect, if any, of Emp gene deletion on definitive hematopoiesis, livers of +/+, +/−, and −/− embryos at E15.5 were examined after H&E and Giemsa staining of paraffin-embedded serial sections, and cytospins. We found few mature erythroid cells in the sinusoids of homozygotes, in contrast to those of either wild-type or heterozygotes, where abundant enucleated red blood cells were observed. Although nucleated erythrocytes were found in both wild-type and mutant embryos, their relative proportions were very different: the less mature forms (proerythroblasts) predominated in the −/− embryos whereas the more mature forms (polychromatophilic/orthochromatic and enucleated erythrocytes) were most common in +/+ and +/− embryos. Furthermore, erythroblastic islands consisting of a central macrophage surrounded by developing erythroblasts were seen in the cytospin preparations of wild-type and heterozygote livers but not in those of homozygous null livers. Since fetal liver macrophages (FLMs) are indispensable for definitive erythropoiesis, we investigated the effect that Emp’s absence might have on development of FLMs. The E15.5 fetal liver sections were stained with the macrophage-specific F4/80 antigen. Numerous F4/80-positive macrophages were present throughout the liver of normal embryos whereas, the number was substantially reduced in Emp (−/−) liver. In summary, in the absence of Emp, FLMs are significantly reduced and terminal maturation of erythroid cells is negatively affected. Thus, the availability of Emp(−/−) embryos will provide a unique experimental model to study the function of macrophages in definitive erythropoiesis.