Non-globin Protein Research Articles

Forced TR2/TR4 expression in sickle cell disease mice confers enhanced fetal hemoglobin synthesis and alleviated disease phenotypes

Sickle cell disease (SCD) is a hematologic disorder caused by a missense mutation in the adult β-globin gene. Higher fetal hemoglobin (HbF) levels in red blood cells of SCD patients have been shown to improve morbidity and mortality. We previously found that nuclear receptors TR2 and TR4 repress expression of the human embryonic ε-globin and fetal γ-globin genes in definitive erythroid cells. Because forced expression of TR2/TR4 in murine adult erythroid cells paradoxically enhanced fetal γ-globin gene expression in transgenic mice, we wished to determine if forced TR2/TR4 expression in a SCD model mouse would result in elevated HbF synthesis and thereby alleviate the disease phenotype. In a "humanized" sickle cell model mouse, forced TR2/TR4 expression increased HbF abundance from 7.6% of total hemoglobin to 18.6%, accompanied by increased hematocrit from 23% to 34% and reticulocyte reduction from 61% to 18%, indicating a significant reduction in hemolysis. Moreover, forced TR2/TR4 expression reduced hepatosplenomegaly and liver parenchymal necrosis and inflammation in SCD mice, indicating alleviation of usual pathophysiological characteristics. This article shows that genetic manipulation of nonglobin proteins, or transcription factors regulating globin gene expression, can ameliorate the disease phenotype in a SCD model animal. This proof-of-concept study demonstrates that modulating TR2/TR4 activity in SCD patients may be a promising therapeutic approach to induce persistent HbF accumulation and for treatment of the disease.

Design of a Novel Heme Protein with a Non-Heme Globin Scaffold

A binding site for iron protoporphyrin IX (heme) was designed and embedded in a photosynthetic non-heme protein, phycocyanin, which forms a globin-like backbone structure, called a globin fold, but lacks sequence similarity to the globin family containing myoglobins and hemoglobins. Based on the structural alignment of the phycocyanin and myoglobin molecules, the proximal and distal His residues were repositioned in the phycocyanin sequence for heme ligation. The heme-binding pocket was created around the His residues by several residue replacements in the phycocyanin core. The synthesized phycocyanin variant, designated as HPY, bound one heme per protein molecule and showed spectroscopic features characteristic of six-coordinated heme proteins. The heme-binding HPY exhibited redox activity with an electrochemical midpoint potential of -130 mV against the standard hydrogen electrode, which was approximately 200 mV lower than the potential of natural myoglobins but 50 mV higher than the typical values of designed heme proteins with four-helix bundle or globin scaffolds. HPY also displayed native-like folding properties, in contrast to these designed heme proteins. However, the bound ferrous heme of HPY was quickly reoxidized by air and did not stably bind O(2), unlike the natural globins. The present results demonstrated that the globin fold of a non-globin protein is suitable for binding heme but is not sufficient for the reversible O(2) binding and myoglobin functions. The comparison of HPY with the natural globins may yield new insights into the essential features for realizing the natural heme protein functions.

Transfusion-dependent congenital dyserythropoietic anaemia with intraerythroblastic inclusions of a non-globin protein.

The congenital forms of dyserythropoiesis comprise a group of hereditary disorders characterized by ineffective erythropoiesis as the predominant mechanism of anaemia and morphologically abnormal erythroblasts. Up to now three major forms and four variants have been described. Group VII is characterized by dyserythropoiesis with intraerythroblastic precipitation of a non-globin protein. Here we described a case of dyserythropoietic anaemia presenting neonatally and requiring regular blood transfusions. Optical and electronic microscopy studies confirmed that this case was very similar to those in two previously reported transfusion-dependent patients with an unusual type of congenital dyserythropoietic anaemia.

A (re)initiation‐dependent cell‐free protein‐synthesis system from mouse erythroleukemia cells

Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis. When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post-mitochondrial supernatants show a high in vitro protein-synthesis activity. Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine. Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells. This reduction of the protein-synthesizing capacity is also observed in vivo. Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non-globin protein synthesis is reduced and globin is preferentially synthesized. Haemin (40 microM) as well as purified eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin-controlled eIF-2 alpha kinase. This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.

Prenatal diagnosis of thalassemia major by fetal blood analysis: experience with 1000 cases.

In this report we have summarized our experience with the prenatal diagnosis of beta-thalassemia in 1000 pregnancies followed at least until 12 months after birth. In the majority of these cases, the thalassemia lesion was the nonsense mutation at the codon corresponding to amino acid 39, which produces the hematological phenotype of beta o-thalassemia. Fetal blood sampling was carried out by placental aspiration, by which a sufficient amount of fetal blood for analysis was obtained in the majority of cases (99 per cent). The fetal mortality associated with fetal blood sampling was 6.3 per cent. Those placental samples contaminated by maternal cells were successfully purified by Orskov lysis. Fetal blood was analysed by globin chain synthesis on CM-52 columns, which gave reliable results. Two misdiagnoses (0.2 per cent) have been made of which one was due to a non-globin protein co-migrating with the beta-chains while the other resulted from a misclassification of the type of thalassemia segregating in the family.

Antenatal diagnosis of thalassemia major in Sardinia.

In this report, we summarized our experience, carried out in Sardinia, with antenatal diagnosis in one thousand pregnancies in which the fetus was at risk for homozygous beta-thalassemia. In the majority of these cases, the thalassemia lesion segregating in the family was the nonsense mutation at the codon corresponding to amino-acid 39. At the outset (976 cases) we used globin chain synthesis analysis by column chromatography on fetal blood obtained by placental aspiration, and recently (24 cases) we employed the synthetic oligonucleotide method on amniocyte DNA. Apart from 126 pregnancies still in progress, in all the other cases the diagnosis has been confirmed. In the majority of the cases (99%), we obtained sufficient fetal blood for the analysis. The fetal mortality associated with placental aspiration was 6.1%. The biochemical analysis gave reliable results. We had two misdiagnoses (0.2%): one due to a nonglobin protein comigrating with the beta chains and the other for a misclassification of the type of thalassemia segregating in the family. The oligonucleotide method gave clear-cut results in all the cases tested. The method was sensitive enough to detect the mutation directly in the DNA isolated from 20-25 ml of amniotic fluid in 75% of the pregnancies tested. In one case, we successfully employed this method for the analysis of the DNA isolated from chorionic villi. The oligonucleotide method seems to be the best procedure for monitoring the pregnancies at risk for beta-thalassemia in places where one or a few beta-thalassemia lesions are prevalent.

Cloned cDNA for rabbit erythrocyte carbonic anhydrase I: A novel erythrocyte-specific probe to study development in erythroid tissues.

Present understanding of gene expression in erythropoietic tissues is derived solely from studies of the globin genes. Of the three distinct carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) isozymes, carbonic anhydrase I is erythrocyte-specific and, in humans, is under developmental control. The appearance of carbonic anhydrase I in the erythrocyte late in fetal life follows closely the gamma- to beta-globin switch. In order to study the expression of this erythrocyte-specific nonglobin protein, we set out to isolate a cloned carbonic anhydrase I cDNA. A mixture of 17-base-long synthetic oligonucleotides was used as an in situ hybridization probe to screen a rabbit reticulocyte cDNA library. Two clones were isolated, and the complete nucleotide sequence of the clone with the largest insert was determined and shown to code for carbonic anhydrase I. This clone, designated pRCAI, is near full length and has provided the 40% of the amino acid sequence of rabbit carbonic anhydrase I, which was not known hitherto. The deduced primary structure has revealed potentially significant changes in the vicinity of the active site of the rabbit carbonic anhydrase I when compared with carbonic anhydrase I and II sequences from other species.

Changes in template activity of protein and globin mRNA during Chironomus development

A wheat-germ cell-free protein synthesizing system was established that efficiently translates fourth instar mRNA from Chironomus thummi. The translation products were analyzed by double immunoprecipitation with specific antibodies against Chironomus hemoglobins. The predominant translation products were shown to be globins, comprising about 50% of total protein synthesized. Two globins, globins 2 and 3, which are specific for the fourth instar in vivo, constitute most of the globin produced. The wheatgerm system translates also efficiently total cytoplasmic RNA, purified from animals at successive developmental stages. The kinetics of protein synthesis during development indicate that Chironomus mRNA template activity is low during larval molting and at metamorphosis. RNA from intermolt larvae generally demonstrates high template activity. In the fourth instar two distinct peaks of activity are resolved, one associated with newly molted fourth instars and a second one associated with the prepupal stage. These RNAs direct the synthesis mostly of non-globin proteins. Immunoprecipitation of the translation products shows that globin synthesis is high in intermolt larvae decreasing to very low, or background levels in larvae molting or metamorphosing to pupae. Globins 2 and 3 are synthesized exclusively by newly synthesized globin transcripts in the fourth instar stage. The results show stage-specific translational activity of globin mRNAs during Chironomus development and suggest hormonal control of globin production.

Modulation of bone marrow heme and protein synthesis by trace elements

We examined the effect of heavy metals and trace elements, such as Cu, Au, Cd, Zn, and Pb, on heme synthetic and degradatory enzymes in rat bone marrow suspensions. Cu, Cd, and Au, at a low concentration of 5 × 10 −6 m, inhibited δ-aminolevulinic acid (δALA) dehydratase by 61, 47, and 47% respectively. A similar concentration of Zn did not inhibit the enzyme activity and, in fact, was able to protect against Au and Pb inhibition in the normal marrow. Cd was unable to recover any of the enzyme activity and, in fact, caused further inhibition. We determined the rate of cellular protein synthesis by measuring the rate of incorporation of radiolabeled precursor [ 14C]leucine. Cu and Au, at a concentration of 10 −5 m, inhibited leucine incorporation into marrow globin and nonglobin protein by 26 and 34%, respectively. Substantially higher concentrations of Zn, as well as Fe, were required to inhibit leucine incorporation into protein. These studies led us to postulate that the inhibition of δALA dehydratase by heavy metals was directly related to the inhibition of heme and protein synthesis in the bone marrow cell suspensions. No evidence of a significant change in heme oxygenase activity in vitro was observed after treatment with any of the heavy metals or trace elements used. These results suggest that trace elements by themselves or in a combination reduce the capacity of the erythroid cell to synthesize heme, the prosthetic group of many hemoproteins. In addition, we have shown that in an iron-overloaded rat the bone marrow contains approximately 45% more heme oxygenase activity than that of the control. Administration of Au alone did not increase heme degradation, but the combination of Fe and Au resulted in a marked induction of heme oxygenase. Finally, for the first time we were able to demonstrate that excess iron and/or decreased Zn concentration may predispose δALA dehydratase to inhibition by an interaction with other metals, such as Pb, by an undefined mechanism(s).

Proteins synthesized by rabbit reticulocyte membrane-bound ribosomes

Rabbit reticulocyte membrane-bound ribosomes liberated by deoxycholate treatment contain degraded forms of ribosomal and messenger RNA. This degradation occurs after the liberation of the ribosomes from the membranes by the detergent because intact birosomal and messenger RNA can be extracted from washed membranes by phenol treatment. Increasing the ionic strength of the detergent buffer prevents this RNA degradation and allows the recovery of membrane-bound ribosomes capable of protein synthesis. Comparison of the proteins synthesized in vitro by the polyribosomes shows that the main protein produced by both free and membrane-bound ribosomes is globin. However, the two types of polyribosomes could be distinguished by the nonglobin proteins they produce.

Sucrose density gradient analysis of erythrocyte membranes in hemolytic anemias

To investigate the membrane abnormalities that may play a pathophysiologic role in several hemolytic anemias we determined the density distribution on sucrose density gradients of human red blood cell (RBC) membranes from patients with these disorders, from normal controls, and from incubated normal RBC. We analyzed the fractions for membrane-adsorbed hemoglobin (Hb), globin, and nonglobin cytoplasmic proteins. The relationship between the cytoplasmic proteins adsorbed on the membranes and the specific gravity (SG) of the membranes was linear. An increase in SG of the entire membrane population was seen in Hb C disease due to adsorbed Hb. Subpopulations of membranes with increased SG due to adsorption of nonglobin protein were evident in the membranes from two splenectomized patients with hemolytic glucose-6-phosphate dehydrogenase (G6PD) variants. Dense membrane subpopulations found in RBC membranes from three splenectomized patients with Hb Köln were associated with adsorbed globin, while similar subpopulations in RBC membranes from three splenectomized patients with hereditary spherocytosis demonstrated increased SG due to adsorbed Hb. Splenectomized normals had no such abnormality in membrane density. Sucrose density gradients demonstrate that membrane bound cytoplasmic protein is characteristic of the RBC membranes in several hemolytic disorders. Additionally, gradients are useful for the isolation and further analysis of those subpopulations of RBC membranes with abnormal SG and exaggerated membrane protein abnormalities.

Sucrose Density Gradient Analysis of Erythrocyte Membranes in Hemolytic Anemias

To investigate the membrane abnormalities that may play a pathophysiologic role in several hemolytic anemias we determined the density distribution on sucrose density gradients of human red blood cell (RBC) membranes from patients with these disorders, from normal controls, and from incubated normal RBC. We analyzed the fractions for membrane-adsorbed hemoglobin (Hb), globin, and nonglobin cytoplasmic proteins. The relationship between the cytoplasmic proteins adsorbed on the membranes and the specific gravity (SG) of the membranes was linear. An increase in SG of the entire membrane population was seen in Hb C disease due to adsorbed Hb. Subpopulations of membranes with increased SG due to adsorption of nonglobin protein were evident in the membranes from two splenectomized patients with hemolytic glucose-6-phosphate dehydrogenase (G6PD) variants. Dense membrane subpopulations found in RBC membranes from three splenectomized patients with Hb Köln were associated with adsorbed globin, while similar subpopulations in RBC membranes from three splenectomized patients with hereditary spherocytosis demonstrated increased SG due to adsorbed Hb. Splenectomized normals had no such abnormality in membrane density. Sucrose density gradients demonstrate that membrane bound cytoplasmic protein is characteristic of the RBC membranes in several hemolytic disorders. Additionally, gradients are useful for the isolation and further analysis of those subpopulations of RBC membranes with abnormal SG and exaggerated membrane protein abnormalities.

An analysis of hemoglobin synthesis in erythropoietic cells

Newts ( Triturus cristatus) rendered anemic by phenylhydrazine exhibit a delayed erythropoietic response in the circulating blood, consisting of successive waves which develop as homogeneous erythroid populations corresponding to the different developmental stages. The initial wave was the object of quantitative study of hemoglobin synthesis and accumulation in vivo, using [ 3H]leucine incorporation into hemoglobin as a method. Correlative studies showed that: (1) Incorporation of [ 3H]leucine into hemoglobin increased by an identical proportion over time in all developmental stages and, when corrected for declining specific activity of intracellular leucine, was linear throughout the 5-hr labeling period in vivo; (2) the specific radioactivities of intracellular soluble leucine (and of tRNA Leu) were virtually identical in early and late stages; and (3) there was no turnover of hemoglobin during erythropoiesis. The rate of hemoglobin synthesis per cell was maximal in early erythroid cells and decreased significantly in subsequent stages. An increasing proportion of protein synthesis in erythroid cells was committed to hemoglobin production during development, an increase which resulted mainly from a proportionately greater decline in production and net accumulation of nonglobin proteins rather than enhanced synthesis of hemoglobin. Despite decreasing cellular levels of hemoglobin synthesis, total hemoglobin-synthesizing capacity, and total hemoglobin content in the erythron (total erythroid cell population) increased during development as a result of mitotic expansion of the erythroid cell population. Pulse-chase experiments conducted either in vivo or in vitro suggested the presence of a pool of globin monomers or dimers whose relative size was insignificant in early erythroid cells but gradually enlarged in subsequent developmental stages. The declining rates of hemoglobin synthesis per cell may reflect decreasing cellular levels of globin mRNA but do not appear to result from decreasing ribosomal availability or altered polyribosomal function.

Structural analysis of the minor human hemoglobin components: Hb AIa1, Hb AIa2 and Hb AIb.

Human hemolysate contains several minor hemoglobin components, including Hb AIa1, Hb AIa2, Hb AIb and Hb AIc which are post-translational modifications of the major component, Hb A0. Hb AIc is known to contain glucose attached to the N terminus of the beta chains by a ketoamine linkage. We separated the alpha and beta globin chains from purified Hb AIa1, Hb AIa2 and Hb AIb by ion-exchange chromatography. The beta chains were reducible by sodium borohydride and gave a positive thiobarbituric acid test. These results indicated that they are modified by ketoamine-linked carbohydrate. In addition, phosphate analysis revealed 1.5 phosphate residue associated with each beta AIa1 chain and 1 phosphate residue with each beta AIa2 chain. Hb AIa1, Hb AIa2 and Hb AIb were all found to be contaminated by non-globin proteins. Protein-sequencing approaches demonstrated that the N termini of beta AIa1, beta AIa2 and beta AIb were blocked. In support of this conclusion, analysis of tryptic digests of beta AIa2 and B AIb revealed modified N-terminal peptides. We conclude that, like Hb AIc, components Hb AIa1, Hb AIa2 and Hb AIb also contain a sugar moiety linked to the N terminus of the beta chain.

Synthesis of non-globin proteins in rabbit-erythroid cells. Synthesis of a lipoxygenase in reticulocytes.

Peripheral rabbit reticulocytes synthesize at least 30 non-globin proteins. One of them is identified as a characteristic lipoxygenase on the basis of its molecular weight, its immunological properties and its behaviour on an ion-exchange column. The enzyme is not produced in bone marrow cells. The synthesis of the lipoxygenase in peripheral blood cells commences on the 3rd day of a bleeding anaemia, increases up to the 5th day and stays constant thereafter at least up to the 14th day. It is concluded that the appearence of the lipoxygenase, which plays a key role in the degradation of mitochondria in the course of maturation of reticulocytes to erythrocytes, is regulated at the translational level.

Studies of the terminal stages of hemoglobin synthesis: I. Stimulation of globin chain synthesis by native hemoglobin subunits

A homologous canine reticulocyte cell-free system was utilized to determine the effects of native hemoglobin subunits on the synthesis of the α and β chains of globin. The addition of either αH or βH in concentrations of 10-3-10-5 M to the cell-free system caused a reproducible increase in synthesis of globin to a maximum of 1½ times that of control values. Tetrameric hemoglobin and other nonglobin proteins in similar concentrations did not produce this effect. While the effect of added αH was to primarily increase β globin synthesis, thus lowering the α/β ratio, βH increased the rates of synthesis of both α and β globin equally. Hemin, which increases protein synthesis but does not change the α/β ratio, was not responsible for the observed changes. These findings suggest that native αH and βH are each independently capable of influencing the rate of globin chain synthesis but do not function by a classical feedback inhibition mechanism. Since the presence of native subunits prolonged the activity of the cell-free system rather than increasing the rate of chain synthesis, the effects may be mediated secondarily by interaction with preformed monomer and dimer units or other components in the system. In contrast to the effects of native hemoglobin subunits, denatured α and β globin, but not denatured hemoglobin or other nonglobin proteins, decreased the rate of synthesis of both α and β globin chains of hemoglobin. This effect may potentiate the genetically defective rate of protein synthesis in the thalassemia syndromes.

Studies of the Terminal Stages of Hemoglobin Synthesis: I. Stimulation of Globin Chain Synthesis by Native Hemoglobin Subunits

A homologous canine reticulocyte cell-free system was utilized to determine the effects of native hemoglobin subunits on the synthesis of the α and β chains of globin. The addition of either αH or βH in concentrations of 10-3-10-5 M to the cell-free system caused a reproducible increase in synthesis of globin to a maximum of 1½ times that of control values. Tetrameric hemoglobin and other nonglobin proteins in similar concentrations did not produce this effect. While the effect of added αH was to primarily increase β globin synthesis, thus lowering the α/β ratio, βH increased the rates of synthesis of both α and β globin equally. Hemin, which increases protein synthesis but does not change the α/β ratio, was not responsible for the observed changes. These findings suggest that native αH and βH are each independently capable of influencing the rate of globin chain synthesis but do not function by a classical feedback inhibition mechanism. Since the presence of native subunits prolonged the activity of the cell-free system rather than increasing the rate of chain synthesis, the effects may be mediated secondarily by interaction with preformed monomer and dimer units or other components in the system. In contrast to the effects of native hemoglobin subunits, denatured α and β globin, but not denatured hemoglobin or other nonglobin proteins, decreased the rate of synthesis of both α and β globin chains of hemoglobin. This effect may potentiate the genetically defective rate of protein synthesis in the thalassemia syndromes.

Globin synthesis in bone marrow cells of patients with sickle cell anemia and beta O-thalassemia: contamination of the beta-chain with non-globin proteins.

Chromatographic separation of labeled globin chains was performed in stroma-free hemolysates prepared from peripheral blood and bone marrow cells of 11 patients with beta O-thalassemia and 2 patients with sickle cell anemia. A small radioactivity peak, slightly preceding the beta-chain and more prominent in bone marrow cells, was often observed. This peak, which represents synthesis of non-globin proteins, did not exceed 5% of the radioactivity incorporated in the alpha-chain. It is concluded that contamination of the beta-chain with non-globin proteins undoubtedly occurs, but its extent is insufficient to explain the different synthetic ratios which have been repeatedly observed in peripheral blood and in bone marrow cells of patients with heterozygous beta-thalassemia.

Homozygous beta0- and beta+ - thalassemia in Kurdish Jews and Arabs.

Chromatographic separation of labeled globin chains was performed on peripheral blood of 38 patients with homorygous β-thalassemia. In 11 patients globin synthesis was also studied in bone-marrow cells. The results showed β/α synthetic ratios in the common range of β+-thalassemia (from 0.11 to 0.39) in 16 out of 27 Kurdish Jews (59%) and in ten out of 11 Arabs (91%). In nine Kurdish Jews (33%) and in one Arab βd`-thalassemia was found. In a few patients from the latter group a very small peak was detected in the β region, which co-chromatographed with β-chains from non-thalassemic individuals, but apparently represents synthesis of a non-globin protein. In two affected siblings, synthesis of small amounts of β-chains was demonstrated in peripheral blood (β/α 0.018–0.025) and in one sample of bone-marrow cells (β/α 0.046). These ratios are lower than any previously reported in β+-thalassemia. Identification of the presumed β-peak in these patients was based on an approximate tenfold increase in β/α, γ/α an...

Control of globin synthesis by hemin. Regulation by hemin of the formation and inactivation of a translational repressor of globin synthesis in rabbit reticulocyte lysates

Considerable evidence indicates that the control of protein synthesis by hemin in rabbit reticulocytes and lysates is mediated by a translational repressor which inhibits polypeptide initiation. Hemin appears to prevent the formation of this inhibitor by binding directly to its precursor, the pro-inhibitor. Hemin also binds to other non-globin proteins in the lysate, and the level of hemin required to suppress the formation of the inhibitor decreases as the pro-inhibitor is purified. Globin or apomyoglobin can prevent or reverse the effect of hemin on the inhibitor, but human serum albumin can only partially prevent and cannot reverse this hemin action. Hemin inactivates the inhibitor by interacting directly with it rather than by blocking its action on the ribosomes. Finally, this report establishes that the hemin-sensitive inhibitory activity formed shortly after hemin deprivation and the hemin-resistant inhibitory activity formed after longer periods do have a precursor-product relationship. This strengthens the concept that repressor formation involves a sequence of conformational changes producing inhibitory activities which have different properties at different stages.