The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact.

Although the association of erythroblasts with macrophages has been well documented in the human bone marrow, the function and identification of the intimate contacts occurring between the membranes of these two cell types in the physiology of erythropoiesis is not known. Using in vitro cultures of human peripheral blood derived erythroid progenitors, we have shown the presence of erythroblastic islands consisting of a central macrophage surrounded by a ring of erythroblasts that undergo terminal maturation leading to enucleation. However, when cultures were carried in the absence of intact macrophages, erythroid cells matured to the late erythroblast stage but failed to enucleate. Furthermore, the number of erythroid cells was markedly reduced in macrophage-depleted cultures, suggesting that the erythroblast-macrophage contact promotes proliferation and terminal maturation of erythroid cells leading to their enucleation. To examine the molecule(s) involved in the interaction between erythroblasts and macrophages, we have used a cell attachment assay involving incubation of solubilized surface-labeled erythroblasts with macrophage membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Erythroblast surface proteins specifically attached to a 30-kD protein from macrophage membranes, whereas no adhesion was seen to the protein standards. An apparently similar protein of 30 kD was also detected on erythroblasts and was shown to mediate erythroblast-erythroblast contact in addition to the erythroblast-macrophage contact. The extraction of plasma membranes with Triton X-100 showed that the 30-kD protein is linked to the membrane skeleton via an integral membrane protein both in erythroblasts and macrophages. Furthermore, our results show that the cell:cell interactions mediated by the 30-kD protein are calcium-independent and could be specifically inhibited by heparin. We conclude that the association of erythroblasts with macrophages promotes erythroid proliferation and maturation leading to erythroblast enucleation and that a 30-kD heparin-binding protein present on the surface of macrophages and erythroblasts is involved in this contact. This protein is capable of binding homotypic and heterotypic cells.

Molecular basis of spectrin deficiency in hereditary pyropoikilocytosis.

Hereditary pyropoikilocytosis (HPP) is a recessively inherited hemolytic anemia characterized by severe poikilocytosis and red blood cell fragmentation. HPP red blood cells are partially deficient in spectrin and contain a mutant alpha or beta-spectrin that is defective in terms of spectrin self-association. Although the nature of the latter defect has been studied in considerable detail and many mutations of alpha-spectrin and beta spectrin have been identified, the molecular basis of spectrin deficiency is unknown. Here we report two mechanisms underlying spectrin deficiency in HPP. The first mechanism involves a thalassemia-like defect characterized by a reduced synthesis of alpha-spectrin as shown by studies involving synthesis of spectrin in two unrelated HPP probands and their parents: One parent carries the elliptocytogenic spectrin mutation, whereas the other parent is fully asymptomatic. Peripheral blood mononuclear cells as a source of erythroid burst-forming unit (BFUe) were cultured in a two-phase liquid culture system that gives rise to terminally differentiated erythroblasts. Pulse-labeling studies of an equal number of erythroblasts or morphologically identical maturity showed that the synthesis of alpha-spectrin as well as the mRNA levels as measured by the competitive polymerase chain reaction (PCR) method are markedly reduced in the presumed asymptomatic carriers and the HPP probands. In contrast, the synthesis and mRNA levels of beta-spectrin were normal. These results constitute a direct demonstration of an alpha-spectrin synthetic defect in a subset of asymptomatic carriers of HPP and HPP probands. The second mechanism underlying spectrin deficiency involves increased degradation of mutant spectrin before its assembly on the membrane. This is evidenced by pulse labeling studies of erythroblasts from a patient with HPP associated with a homozygous state for spectrin alpha I/46 mutation (leu-pro mutation at AA 207 of alpha-spectrin). These studies showed that although spectrin is synthesized in the cytosol in normal amounts, the rate of turnover of alpha-spectrin is faster resulting in about 40% to 50% reduced assembly of alpha-spectrin and beta-spectrin on the membrane. Thus, spectrin deficiency in this case is at least in part caused by increased susceptibility of the mutant spectrin to degradation before its assembly on the membrane. We conclude that at least two separate mechanisms underlie the molecular basis of spectrin deficiency in HPP.

The expression and synthesis of the band 3 protein initiates the formation of a stable membrane skeleton in murine Rauscher-transformed erythroid cells.

While the temporal sequences of the synthesis and assembly of membrane skeletal proteins has been studied during erythroid maturation, relatively little is known about the events which initiate the assembly of membrane skeleton at the early stages of mammalian erythroid commitment. To investigate the early events that initiate the assembly of the membrane skeleton in mammalian erythroid cells, we have studied the synthesis and assembly of membrane skeletal proteins in murine Rauscher erythroleukemia virus-transformed cells. These cells are blocked in differentiation at around the early progenitor (burst forming unit-erythroid, BFUe) cell stage but can be induced to differentiate in vitro. Pulse-labeling studies reveal that Rauscher cells actively synthesize alpha spectrin, beta spectrin, ankyrin and band 4.1 proteins. However, the synthesis of the band 3 protein and its mRNA are barely detectable in these cells. The peripheral membrane skeletal components assemble only transiently in the membrane skeleton and turn over rapidly, resulting in about 20-fold lower steady state levels than are found in mature erythrocytes. Upon induction with erythropoietin and dimethyl sulfoxide, the mRNA level and synthesis of band 3 are increased about 50-fold. In contrast, the synthesis of spectrin, ankyrin and band 4.1 is increased only about 1.5 to 2.0-fold. However, after induction, the fraction of these proteins assembled on the membrane is increased, their half-lives on the membrane are nearly doubled with a concomitant 4 to 5-fold increase in their steady-state levels. These results suggest that the synthesis of peripheral membrane proteins is detected at the earliest stages of erythroid commitment and increases only slightly during further differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Asynchronous synthesis of membrane skeletal proteins during terminal maturation of murine erythroblasts.

To study the changes in the synthesis of the major membrane skeletal proteins, their assembly on the membrane, and their turnover during terminal red blood cell maturation in vivo, we have compared early proerythroblasts and late erythroblasts obtained from the spleens of mice at different times after infection with the anemia-inducing strain of Friend virus (FVA). Metabolic labeling of these cells indicates striking differences between early and late erythroblasts. In early erythroblasts, spectrin and ankyrin are synthesized in large amounts in the cytosol with proportionately high levels of spectrin and ankyrin messenger RNA (mRNA). In contrast, only small amounts of these polypeptides are incorporated into the skeleton, which is markedly unstable. In late erythroblasts, however, the synthesis of spectrin and ankyrin and their mRNA levels are substantially reduced, yet the net amounts of these polypeptides assembled in the membrane skeleton are markedly increased, and the membrane skeleton becomes stable with no detectable protein turnover. The mRNA levels and the synthesis of the band 3 and 4.1 proteins are increased considerably in terminally differentiated normoblasts with a concomitant increase in the net amount and the half-life of the newly assembled spectrin and ankyrin. Thus, the increased accumulation of spectrin and ankyrin at the late erythroblast stage is a consequence of an increased recruitment of these proteins on the membrane and an increase in their stability rather than a transcriptional upregulation. This is in contrast to band 3 and 4.1 proteins, which accumulate in direct proportion to their mRNA levels and rates of synthesis. These results suggest a key role for the band 3 and 4.1 proteins in conferring a long-term stability to the membrane skeleton during terminal red blood cell differentiation.

Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin.

While varying degrees of spectrin deficiency have been found in the majority of patients with hereditary spherocytosis (HS), a combined severe deficiency of both spectrin and the spectrin-binding protein, ankyrin, has been reported only in two patients with severe HS. To elucidate the molecular basis of these protein deficiencies, we have studied the synthesis, assembly, and the mRNA levels of spectrin and ankyrin in peripheral blood reticulocytes in one of the previously reported probands. Pulse-labeling studies showed that in HS reticulocytes, the synthesis of alpha-spectrin was comparable with control reticulocytes while that of beta-spectrin was increased about fourfold, presumably reflecting increased erythropoietic drive. On the HS reticulocyte membrane, the amount of newly assembled spectrin was reduced to about half of the control values, presumably reflecting a decrease in the synthesis of the spectrin binding protein, ankyrin: the ankyrin synthesis was nearly absent in the cytosol and the amounts of membrane-associated ankyrin were reduced to about half of the normal values. The changes in the amounts of spectrin and ankyrin mRNAs quantitated by slot blot and Northern blot analyses were comparable with changes in the synthesis of these proteins: The alpha spectrin mRNA was within a control range and the beta-spectrin mRNA was slightly increased, while the amounts of ankyrin mRNA were reduced to about 50% of control values. We conclude that the primary defect underlying the combined spectrin and ankyrin deficiency is a deficiency of ankyrin mRNA leading to a reduced synthesis of ankyrin which, in turn, underlies the decreased assembly of spectrin on the membrane.

Detection of protease inhibitors using substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A technique is described to detect the activity of protease inhibitors present in sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAG) containing a copolymerized enzyme substrate. The method involved (1) incorporation of substrate (gelatin or casein) into the SDS-PAG at the time of casting; (2) electrophoresis of the protease inhibitors in the presence of SDS; (3) removal of SDS by washing the gel in 2.5% (w/v) Triton X-100; (4) incubation of the gels in a solution containing the proteolytic enzyme at 37 degrees C for 16 h; and (5) staining undigested substrate with amido black. Standard inhibitors such as bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (SBTI), alpha 1-antitrypsin inhibitor, and a protease inhibitor derived from human articular cartilage have been examined by this method and displayed sharp inhibition bands when the gels were treated with bovine trypsin, chymotrypsin, or other enzymes. The technique cannot be used for precise quantification of protease inhibitors. However, there is a relationship between the concentration of inhibitor used and the intensity of staining. By this means, it was possible to estimate the smallest amount of inhibitor that could be detected (against a particular enzyme) under a given set of conditions. Inhibition was detected when 10 ng of SBTI or 20 ng of BPTI were applied to the gels; human alpha 1-protease inhibitor could be detected at a level of 2-3 micrograms. The technique was used to investigate the effectiveness of the human cartilage inhibitor against a variety of proteolytic enzymes, including thermolysin, Pronase, neutral protease, elastase, protease VII, pepsin, bacterial collagenase, protease IV, and papain.

Purification of phosphofructokinase from rabbit skeletal muscle based on its ability to aggregate in absence of its effectors.

Rabbit muscle phosphofructokinase was purified to homogeneity based on its property to form large aggregates with time at high concentration of its protein in absence of its effectors. The method involves no heat step or treatment with organic solvent or any ion-exchange columns. The enzyme thus prepared, however, exhibits the same kinetic properties as the enzyme purified by more drastic methods.