Reticulocyte Protein Research Articles

Tracking immature reticulocyte proteins for improved detection of recombinant human erythropoietin (rhEPO) abuse.

Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.

A novel recombinant chemokine protein TAT-SDF-1/54-KDEL for blocking CXCR4: bioinformatics analysis and prokaryotic expression (CCR5P.263)

Abstract To explore the potential application of SDF-1/54 mutant in Immunology, a prokaryotic vector pTAT-HA was exerted to express a novel recombinant protein ,TAT/SDF-1/54/KDEL, which contains three distinct functional domains: murine SDF-1/54, a 11-peptide TAT (47-57) derived from human immunodeficiency virus tat trans-activator protein and an endoplasmic reticulum retention four-peptide sequence KDEL.Bioinformatics analysis showed that the estimated half-life of the protein in mammalian reticulocytes,yeast,Escherichia coli in vitro are 2.8h, 10h and 2min. Respectively.Endoplasmic reticulum distribution and localization ratio is 13.0%. The data also shows the novel protein on the secondary structure and topology proteins , protein-binding sites, the three-dimensional map and a possible prediction catalytic pocket and the binding sites. Our expression experiment showed that the expected band of the protein appeared in the precipitate by SDS-PAGE electrophoresis. Western blot confirmed the specificity of the immune response of this protein to the rabbit anti-His-probe antibody.These suggest that the bioinformatics analysis for TAT/54R/KDEL might be an effective approach to predict its structure and functional expression. The expression of the protein in pTAT-HA prokaryotic vector is in the form of inclusion bodies. It offered a new method targeting for CXCR4 implicated immunity regulation,such as HIV, WHIM syndrome, cancer and stem cells mobilization and so on.

The RNA in reticulocytes is not just debris: It is necessary for the final stages of erythrocyte formation

Reticulocytes contain both RNA and micro-organelles and represent the last stage of erythropoiesis before full maturation to red blood cells (RBCs). Even though there is continuing synthesis of hemoglobin and membrane-bound proteins in reticulocytes, the small amount of RNA that they contain has been regarded as non-functional residual material. Here we show that this residual RNA is both functional and essential for further reticulocyte maturation. Reticulocytes from which the remnant RNA had been removed by exposure to RNase did not survive or mature into RBCs in either humans or mice. Conversely, reticulocytes treated with an RNase Inhibitor were able to form normal biconcave cells. Similarly, poor survival was also seen in reticulocytes in which protein synthesis had been blocked. To identify the signaling pathways involved we isolated RNAs in reticulocytes versus those present in fully matured erythroblasts cultured from hematopoietic stem cells. RNAs found in erythroblasts were related to exocytosis, metabolism, and signal transduction all of which are critical for maturation through reticulocyte and into a fully mature, biconcave erythrocyte. Our results suggest that the mRNA in reticulocytes has to be translated into novel proteins that act to preserve mitochondria and maintain cell membrane integrity as reticulocytes mature. These results enhance our understanding of the final stage of erythropoiesis and may clarify why in vitro-generated reticulocytes for transfusion purposes survive poorly.

The Cystic Fibrosis Transmembrane Conductance Regulator Is Regulated by a Direct Interaction with the Protein Phosphatase 2A

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aalpha and B'epsilon and find that the B'epsilon subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies.

Tetrahydrobiopterin depletion and ubiquitylation of neuronal nitric oxide synthase

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N G-nitro- l-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation. Since tetrahydrobiopterin is known to bind near heme and confers stability to the active dimeric structure of nNOS, we wondered if the loss of tetrahydrobiopterin could be an endogenous signal for nNOS ubiquitylation and degradation. We show here in HEK293 cells stably transfected with nNOS that depletion of tetrahydrobiopterin by treatment with 2,4-diamino-6-hydroxypyrimidine leads to destabilization of the dimeric form and enhances ubiquitylation of nNOS. Sepiapterin, a precursor to tetrahydrobiopterin in the salvage pathway, completely reverses the effect of 2,4-diamino-6-hydroxypyrimidine on nNOS ubiquitylation. Consistent with that found in cells, the in vitro ubiquitylation of nNOS by reticulocyte proteins decreases when tetrahydrobiopterin is present. Thus, inadequate amounts of tetrahydrobiopterin may lead to a sustained decrease in the steady state level of nNOS that is not readily reversed.

Ubiquitination and degradation of neuronal nitric-oxide synthase in vitro: dimer stabilization protects the enzyme from proteolysis.

It is established that neuronal NO synthase (nNOS) is ubiquitinated and proteasomally degraded. The metabolism-based inactivation of nNOS and the inhibition of heat shock protein 90 (hsp90)-based chaperones, which are known to regulate nNOS, both lead to enhanced proteasomal degradation of nNOS. The mechanism of this selective proteolytic degradation, or in essence how the nNOS becomes labilized and recognized for ubiquitination and subsequent degradation, has not been determined. In the current study, we used a crude preparation of reticulocyte proteins, which contains ubiquitin-conjugating enzymes and the proteasome, to determine how nNOS is labilized. We found that the inactive monomeric heme-deficient nNOS (apo-nNOS) is rapidly degraded in vitro, consistent with the finding that both metabolism-based inactivation and inhibition of hsp90-based chaperones cause the formation of apo-nNOS and enhance its degradation in vivo. In the current study, we discovered that destabilization of the dimeric nNOS, as determined by measuring the SDS-resistant dimer, is sufficient to trigger ubiquitin-proteasomal degradation. Treatment of nNOS with NG-nitro-L-arginine or 7-nitroindazole led to stabilization of the dimeric nNOS and decreased proteasomal degradation of the enzyme, consistent with that observed in cells. Thus, it seems that the dimeric structure is a major determinant of nNOS stability and proteolysis.

Screening for iron deficiency.

1. Ann Chen Wu, MD* 2. Leann Lesperance, MD, PhD* 3. Henry Bernstein, DO*† 1. *Pediatric Health Associates, Hunnewell Ground Children’s Hospital 2. †Associate Professor of Pediatrics, Harvard Medical School, Boston, MA After completing this article, readers should be able to: 1. Determine the most common cause of iron deficiency in the United States. 2. Describe the pathogenesis of iron deficiency. 3. List populations at high risk for iron deficiency. 4. Outline the common signs and symptoms of iron deficiency. 5. Specify the American Academy of Pediatrics recommendations for screening for iron deficiency. In the March and April issues of Pediatrics in Review, we published a two-part article on managing anemia in a pediatric office practice. This article expands on the various tests for iron deficiency, including some relatively new ones. These articles should be read as complementary.—RJH Iron deficiency is the most common nutritional deficiency in the world, responsible for a staggering amount of ill health, lost productivity, and premature death. Although its prevalence in the United States has declined since the late 1960s, iron deficiency with or without anemia still is seen frequently in infants, toddlers, adolescent females, and women of childbearing age. In fact, iron deficiency anemia remains the most common hematologic disease of infants and children. Anemia is defined as a low hemoglobin (Hgb) concentration or red blood cell (RBC) mass compared with age-specific norms. Anemia may be caused by decreased RBC production, increased RBC destruction, or blood loss. Based on the size of the RBC, hematologists categorize anemia as macrocytic, normocytic, or microcytic. Iron is found in different compartments within the body. Total body iron (measured by ferritin), transport iron (measured by transferrin saturation), serum iron, and other hematologic and biochemical markers are used to describe the degrees of iron deficiency. Iron depletion refers to the earliest stage of diminishing iron stores in the setting of insufficient iron supply. Iron deficiency (without anemia) develops as these iron stores are depleted further and begin to impair Hgb synthesis. Finally, iron deficiency anemia results …

Induced synthesis of albumin-like protein in damaged rat reticulocytes.

In this study, we present evidence that red blood cell (RBC) membrane p68 in the Belgrade (b/b) rat is similar if not identical to rat serum albumin (RSA). Structural homology between RSA and the RBC p68 has been determined by a variety of biochemical and immunological criteria. This albumin-like protein is a normal constituent of rat RBC and it is partially exported by exosomes during erythroid differentiation. The endogenous origin of rat RBC albumin-like protein was demonstrated by monitoring protein synthesis in reticulocytes and by identification of reticulocyte mRNA for albumin. Haemolytic anaemia, either hereditary (b/b rat) or drug-induced (phenylhydrazine-treated normal rat), results in increased accumulation of the albumin-like protein in rat RBCs as a result of its induced synthesis.

Identification of Rabbit Reticulocyte E217K as a UBC7 Homologue and Functional Characterization of Its Core Domain Loop

The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1. E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain.

Purification of a 38-kDa Protein from Rabbit Reticulocyte Lysate Which Promotes Protein Renaturation by Heat Shock Protein 70 and Its Identification as δ-Aminolevulinic Acid Dehydratase and as a Putative DnaJ Protein

We reported recently that a rabbit reticulocyte 66-kDa protein (termed RF-hsp 70 by our laboratory and p60 and hop by others) functions as a hsp 70 recycling protein and markedly enhances the renaturation of luciferase by hsp 70 (Gross, M., and Hessefort, S. (1996) J. Biol. Chem. 271, 16833-16841). In this report, we confirm that the ability of RF-hsp 70 to promote the conversion of hsp 70. ADP to hsp 70.ATP, thus enhancing the protein folding activity of hsp 70, is caused by the purified 66-kDa protein and not by a trace DnaJ/hsp 40 protein contaminant. To determine the relationship between RF-hsp 70 and the DnaJ/hsp 40 heat shock protein family, which also enhances protein renaturation by hsp 70, we purified a 38-kDa protein from rabbit reticulocyte lysate based upon its ability to stimulate renaturation of luciferase by hsp 70. Partial amino acid sequencing of this 38-kDa protein has indicated, unexpectedly, that it is the enzyme delta-aminolevulinic acid dehydratase (ALA-D) and that it does not contain detectable sequences corresponding to the DnaJ/hsp 40 protein family. In addition, immunoblot analysis with a polyclonal antibody made to HeLa cell hsp 40 (from StressGen) confirms that our purified ALA-D contains no hsp 40, although hsp 40 is present in relatively crude rabbit reticulocyte protein fractions. Rabbit reticulocyte ALA-D is about as active in converting delta-aminolevulinic acid to porphobilinogen and as Zn2+-dependent as ALA-D purified from other sources. Rabbit reticulocyte ALA-D stimulates the renaturation of luciferase by hsp 70 up to 10-fold at concentrations that are the same as or less than that of hsp 70, and it has no renaturation activity in the absence of hsp 70. The renaturation effect of ALA-D is additive with that of RF-hsp 70 at limiting or saturating concentrations of each, and, unlike RF-hsp 70, ALA-D does not promote the dissociation of hsp 70.ADP in the presence of ATP. The renaturation-enhancing effect of ALA-D may be caused by a region near its carboxyl terminus which has sequence homology to the highly conserved domain of the DnaJ protein family, which is similar to the sequence homology between this domain and a carboxyl-terminal region in auxilin, a DnaJ-like protein that requires this region for its hsp 70-dependent function (Ungewickell, E., Ungewickell, H., Holstein, S. E. H., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L. E., and Eisenberg, E. (1995) Nature 378, 632-635).

Changes in the Expression of the Heme-Regulated eIF-2α Kinase and Heat Shock Proteins in Rabbit Reticulocytes Maturing during Recovery from Anemia

Changes in the expression of the heme-regulated eIF-2α kinase (HRI), heat shock proteins (Hsps, Hsp90, and 70) and their associated cohorts (p60 and p23) were studied in maturing rabbit reticulocytes during recovery from anemia. Reticulocytosis was induced by injection ofN-acetylphenylhydrazine or by phlebotomy from the ear vein, and circulating red blood cells were fractionated on histopaque density gradients. Northern and Western blot analyses indicated that HRI and hsps mRNA and protein content gradually decreased during maturation of reticulocytes into erythrocytes. Reduction in levels of hsps and HRI was also observed when cells of same age group (density) were compared as the animals recovered from the anemia. Low hematocrits correlated with high levels of hsps expression and with increasing hematocrits hsps expression decreased. Under the conditions used to quantify these protein levels, Hsc70 and p60 were detected in erythrocytes of fully recovered animals. Maintenance of Hsc70 and p60 suggests important ongoing roles for these hsps in protecting the structure and function of proteins in erythrocytes lacking transcriptional and translational machinery.

Spermatocytes and round spermatids of rat testis: The difference betweenin vivoandin vitroprotein patterns

During mammalian spermatogenesis meiotic cell division and spermiogenesis occurs. Gene expression during this process is temporally regulated at the transcriptional and translational levels but the mechanisms are not well understood. In this publication we have investigated the synthesis of proteins in vitro to detect the proteins with a high metabolic turnover and to compare them with the in vivo protein map. RNA of spermatocytes and round spermatid cell populations, purified by centrifugal elutriation, and total testis was isolated. The poly A+ mRNA fraction was translated using a rabbit reticulocyte lysate. The translation products were separated by two-dimensional (2-D) gel electrophoresis using nonlinear 3.5-10 immobilized pH gradients for the first-dimensional separation. The gels with 35S-translated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and scanned using a phosphorimager. A highly reproducible and complex protein pattern was obtained using this methodology. Only rat testis messages were translated. Using Melanie 2 software we could compare and detect more than 1000 proteins on 2-D radioactive images. Some changes could be observed in protein expression between the different cell types but they were not statistically significant. The comparison between the 2-D rat testis map and the in vitro translated patterns show no matching between any spots. This result suggests that the post-transcriptional modifications occurring in the reticulocyte system are not the same as those that occur in vivo in the testis. Rabbit reticulocyte proteins were detected by staining PVDF membranes with colloidal gold. Rat testis and reticulocyte patterns were completely different.

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis.

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.

Extensive Sequencing of Tryptic Peptides of a Rabbit Reticulocyte 66-kDa Protein That Promotes Recycling of Hsp 70: HOMOLOGY TO STRESS-RELATED PROTEINS

Trypsinization and sequence analysis of the 66-kDa rabbit reticulocyte protein (RF-hsp 70), shown in the preceding article to function as a recycling protein for hsp 70, demonstrates striking similarity to the transformation-sensitive human protein IEF SSP 3521 (Honoré, B., Leffers, H., Madsen, P., Rasmussen, H. H., Vandekerckhove, J., and Celis, J. E.(1992) J. Biol. Chem. 267, 8485-8491) and mouse extendin (Blatch, G. L., Lassle, M., Takatori, T., Gandhi, T., Kundra, V., and Zetter, B. R.(1995) Proc. Am. Assoc. Cancer Res. 36, 68). The human and mouse proteins share 97% sequence identity, and sequencing of 20 polypeptides (225 residues) from RF-hsp 70 reveals only 10 differences between the rabbit and human proteins and 13 differences between the rabbit and mouse proteins (96 and 94% identity, respectively). In addition, all three proteins are of similar size, and each contains 11 cysteines. These findings strongly suggest that these three proteins are homologs of the same activity. All differences (but one) between the human and mouse proteins occur within the amino-terminal half of the protein, and there is only one difference among 121 sequenced residues between RF-hsp 70 and the human or mouse protein which occurs within the carboxyl-terminal 70% of the molecule. In addition, where partial sequences of RF-hsp 70 and p60, a chick oviduct protein that shows 70% identity to the human protein (Smith, D. F., Sullivan, W. P., Marion, T. N., Zaitsu, K., Madden, B., McCormick, D. J., and Toft, D. O. (1993) Mol. Cell. Biol. 13, 869-876), overlap (a total of 54 residues), RF-hsp 70 and chick p60 show 78% sequence identity. Studies of the initial digestion of RF-hsp 70 by trypsin indicate that it is first converted to 58- and 54-kDa components, each of which is then converted to a 43-kDa polypeptide. This 43-kDa component is located in the human and mouse proteins at position 124 to about 470. It is converted subsequently to a 31-kDa polypeptide by trypsin hydrolysis at position 207. This 31-kDa component is finally split into 17- and 14-kDa polypeptides that are located at positions 208 to approximately 351 and 352 to approximately 470, respectively. The 14-kDa polypeptide is relatively resistant to further digestion with trypsin, and seven tryptic peptides from other parts of RF-hsp 70 contain internal lysine and/or arginine residues (as do several tryptic peptides produced from IEF SSP 3521 and chick p60). Both features may be due to interference with trypsin action by secondary structure in the protein, since trypsinization of reduced and carboxymethylated RF-hsp 70 results in hydrolysis of the 14-kDa polypeptide and reduces the level of peptides that contain internal lysine and/or arginine, although it does not eliminate them.

Control of protein synthesis by hemin. Purification of a rabbit reticulocyte hsp 70 and characterization of its regulation of the activation of the hemin-controlled eIF-2(alpha) kinase.

We have purified a soluble rabbit reticulocyte protein, previously termed the supernatant factor, that reverses the inhibition of protein synthesis in hemin-deficient lysate by promoting the inactivation of the hemin-controlled eIF-2 alpha kinase (HCR) mediating the effect of hemin deficiency. We have identified the supernatant factor as a member of the heat shock protein 70 family, denoted hsp 70(R), based upon its size (72 kDa), specific reaction to a monoclonal antibody against eukaryotic hsp 70, strong binding affinity for ATP, and endogenous ATPase activity. We have investigated the role of hsp 70(R) and hemin in the regulation of the activation of HCR from its latent precursor (ProHCR) and the translational control of protein synthesis in rabbit reticulocyte lysate. We find that autophosphorylation of Pro-HCR is reduced by about 75% by adding saturating hsp 70(R) and almost completely reduced by adding either saturating hemin or limiting hemin plus limiting hsp 70(R). In contrast, autophosphorylation of HCR, which is similar in magnitude to that of ProHCR, is unaffected by adding either saturating hsp 70(R), saturating hemin, or limiting amounts of both. The activation of HCR (measured by inhibition of protein synthesis) from isolated ProHCR is completely prevented by hsp 70(R) in the presence, but not absence, of dithiothreitol. This suppression appears to be due to the association of hsp 70(R) with ProHCR, since hsp 70(R) action is prevented by ATP/Mg2+ and because activation of HCR from less purified ProHCR, that has associated hsp 70(R), is suppressed by dithiothreitol alone. This association is confirmed by sucrose gradient centrifugation, which shows co-sedimentation of some hsp 70(R) with ProHCR following preincubation together that is prevented by ATP/Mg2+ and does not occur after conversion of ProHCR to HCR. Limiting hsp 70(R) reduces the concentration of hemin required to prevent activation of HCR from isolated ProHCR from 0.75 to 0.15 microM and the optimal hemin concentration needed to maintain protein synthesis in reticulocyte lysate from 25 to 10 microM. Limiting hsp 70(R) also allows the delayed addition of hemin to suppress activation of HCR from ProHCR and to reverse inhibition of protein synthesis in hemin deficient lysate. The association of hsp 70(R) with ProHCR also underlies the observation that much more protein is synthesized in reticulocyte lysate in the absence of hemin at 25 degrees C than at temperatures of 30 degrees C or greater. These observed effects may be specific to hsp 70(R), since they are not observed with rabbit reticulocyte eIF-2 or eIF-2B, and since the comparable hsp 70 from bovine brain is incapable of maintaining or restoring protein synthesis in hemin-deficient lysate.

Translation of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3' untranslated region.

During red blood cell differentiation, the mRNA encoding rabbit erythroid 15-lipoxygenase (LOX) is synthesized in the early stages of erythropoiesis, but is only activated for translation in peripheral reticulocytes. Erythroid LOX, which like other lipoxygenases catalyses the degradation of lipids, is unique in its ability to attack intact phospholipids and is the main factor responsible for the degradation of mitochondria during reticulocyte maturation. Strikingly, rabbit erythroid LOX mRNA has 10 tandem repeats of a slightly varied, pyrimidine-rich 19 nt motif in its 3'-untranslated region (3'-UTR). In this study we demonstrate, using gel retardation and UV-crosslinking assays, that this 3'-UTR segment specifically binds a 48 kDa reticulocyte protein. Furthermore, the interaction between the 3'-UTR LOX repeat motif and the 48 kDa protein, purified to homogeneity by specific RNA chromatography, is shown to be necessary and sufficient for specific translational repression of LOX as well as reporter mRNAs in vitro. To our knowledge this is the first case in which translation, presumably at the initiation step, is regulated by a defined protein-RNA interaction in the 3'-UTR.

Distinct fetal Ank-1 and Ank-2 related proteins and mRNAs in normal and nb/nb mice

Mice homozygous for the mutation normoblastosis (gene symbol nb on chromosome 8) are deficient in erythroid ankyrin (ANK-1) and have a severe hemolytic anemia throughout life. Characteristic of the disease is a dramatic decrease in the level of expression of the Ank-1 gene (chromosome 8). The other major ankyrin transcript, brain ankyrin (Ank-2 on chromosome 3) is expressed at normal levels in nb/nb mice. Surprisingly, nb/nb fetuses have normal erythrocyte counts despite the decreased levels of Ank-1 transcripts. We previously hypothesized that fetal-specific ankyrin-related proteins could exist in nb/nb fetuses to account for the lack of detrimental effects of ANK-1 deficiency. In the present report, Western and Northern blot analyses were performed on hematopoietic cells isolated from nb/nb and +/+ fetuses. An ANK-1-related protein (165 Kd) in fetal reticulocytes persisted in adult nb/nb but not in +/+ reticulocytes. An Ank-1-related transcript of 5.5 kb was found in fetal reticulocytes. This transcript appeared to be upregulated in nb/nb but not in +/+ adult reticulocytes. A fetal-specific ANK-2-related protein (155 Kd) was present in nb/nb and in +/+ fetal reticulocytes. Ank-2-related fetal liver mRNAs were present during the time the liver was actively generating erythrocytes. Neither the Ank-2-related transcripts nor the 155-Kd ANK-2-related protein were found in +/+ or mutant adult reticulocytes. The data indicate that (1) unique ankyrin-related proteins and mRNAs present in fetal erythrocytes may stabilize the ankyrin-deficient nb/nb erythrocytes and (2) adult nb/nb mice may upregulate fetal gene transcripts to compensate for the ANK-1 deficiency.

Distinct Fetal Ank-1 and Ank-2 Related Proteins and mRNAs in Normal and nb/nb Mice

Abstract Mice homozygous for the mutation normoblastosis (gene symbol nb on chromosome 8) are deficient in erythroid ankyrin (ANK-1) and have a severe hemolytic anemia throughout life. Characteristic of the disease is a dramatic decrease in the level of expression of the Ank-1 gene (chromosome 8). The other major ankyrin transcript, brain ankyrin (Ank- 2 on chromosome 3) is expressed at normal levels in nb/nb mice. Surprisingly, nb/nb fetuses have normal erythrocyte counts despite the decreased levels of Ank-1 transcripts. We previously hypothesized that fetal-specific ankyrin-related proteins could exist in nb/nb fetuses to account for the lack of detrimental effects of ANK-1 deficiency. In the present report, Western and Northern blot analyses were performed on hematopoietic cells isolated from nb/nb and +/+ fetuses. An ANK-1- related protein (165 Kd) in fetal reticulocytes persisted in adult nb/nb but not in +/+ reticulocytes. An Ank-1-related transcript of 5.5 kb was found in fetal reticulocytes. This transcript appeared to be upregulated in nb/nb but not in +/+ adult reticulocytes. A fetal- specific ANK-2-related protein (155 Kd) was present in nb/nb and in +/+ fetal reticulocytes. Ank-2-related fetal liver mRNAs were present during the time the liver was actively generating erythrocytes. Neither the Ank-2-related transcripts nor the 155-Kd ANK-2-related protein were found in +/+ or mutant adult reticulocytes. The data indicate that (1) unique ankyrin-related proteins and mRNAs present in fetal erythrocytes may stabilize the ankyrin-deficient nb/nb erythrocytes and (2) adult nb/nb mice may upregulate fetal gene transcripts to compensate for the ANK-1 deficiency.

Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.

Hereditary Elliptocytosis Due To Both Qualitative and Quantitative Defects in Membrane Skeletal Protein 4.1

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.