CD47 surface stability is sensitive to actin disruption prior to inclusion within the band 3 macrocomplex

AbstractCD47 is an important ‘marker of self’ protein with multiple isoforms produced though alternative splicing that exhibit tissue-specific expression. Mature erythrocytes express CD47 isoform 2 only, with membrane stability of this version dependent on inclusion within the band 3 macrocomplex, via protein 4.2. At present a paucity of information exists regarding the associations and trafficking of the CD47 isoforms during erythropoiesis. We show that CD47 isoform 2 is the predominant version maintained at the surface of expanding and terminally differentiating erythroblasts. CD47 isoforms 3 and 4 are expressed in all cell types tested except mature erythrocytes, but do not reach the plasma membrane in erythroblasts and are degraded by the orthochromatic stage of differentiation. To identify putative CD47 interactants, immunoprecipitation combined with Nano LC-MS/MS mass spectrometry was conducted on the erythroleukaemic K562 cell line, expanding and terminally differentiating primary erythroblasts and mature erythrocytes. Results indicate that prior to incorporation into the band 3 macrocomplex, CD47 associates with actin-binding proteins and we confirm that CD47 membrane stability is sensitive to actin disrupting drugs. Maintenance of CD47 at the cell surface was also influenced by dynamin, with sensitivity to dynamin disruption prolonged relative to that of actin during erythropoiesis.</jats:p

Differential Proteomic Analysis of Human Erythroblasts Undergoing Apoptosis Induced by Epo-Withdrawal

The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34+ cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis

The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation

Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes

CFTR potentiators partially restore channel function to A561E, a cystic fibrosis mutant with a similar mechanism of dysfunction as F508del-CFTR

BACKGROUND AND PURPOSE: Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(−) channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease. EXPERIMENTAL APPROACH: Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques. KEY RESULTS: Low temperature incubation delivered a small proportion of A561E-CFTR protein to the cell surface. Like F508del-CFTR, low temperature-rescued A561E-CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E-CFTR also exhibited thermoinstability, losing function more quickly than F508del-CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561E-CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561E- and F508del-CFTR Cl(−) channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches. CONCLUSIONS AND IMPLICATIONS: Like F508del-CFTR, A561E-CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561E-CFTR. Transformational drug therapy for A561E-CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability

Non-muscle myosin II drives vesicle loss during human reticulocyte maturation

The process of reticulocyte maturation into fully mature erythrocytes that occurs in circulation is known to be characterised by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodelling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we use high-throughput proteomic methods to highlight differences between erythrocytes, cultured and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) as exhibiting differential abundance and phosphorylation status between reticulocytes and erythrocytes and localize it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with NMII inhibition, we demonstrate involvement of NMIIA in reticulocyte remodelling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation

Proteins with altered abundance in SDL with a change in average ratio of >2 and a <i>t</i>-test of <i>p</i><0.05.

<p>Characterization of proteins fractionated by 2D-PAGE and identified by both the DeCyder software (all gels, independent <i>t</i>-test, change in average ratio of >2 with a <i>t</i>-test of <i>p</i><0.05 ) and Mass Spectrometry, ranked in order of change in average ratio. From the 11 spots up-regulated in SDL, 11 different proteins were identified. Spot No. is number of the spot picked and shown on an SDL 2D gel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone-0038356-g003" target="_blank">Figure 3A</a>); <i>t</i>-test and change in average ratio calculated by the DeCyder 2D Differential Analysis software when comparing the four SDL with the four ESDL 2D gels; Identified Proteins: full name of the protein identified by mass spectrometry, including isoform, as given by NCBI; NCBI Accession number and Gene ID number together with the official symbol provided by HUGO Gene Nomenclature Committee (HGNC) in brackets; Experimental (E) & Theoretical (T) Molecular mass (in kDa) and p<i>I</i>: the experimental (E) molecular mass and p<i>I</i> were determined by eye for each spot picked, whereas the theoretical (T) molecular mass and p<i>I</i> were determined using EditSeq (DNA lasergene 8) on the protein sequence of gi number identified; Number of peptide matched: total number of unique peptides matched to the protein identified; Mascot protein score: the protein score for that gi number is given. Protein score is defined as -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 66 (>66) are considered significant identifications (p<0.05). Where MSMS was performed, the calculated precursor ion mass and resulting peptide sequences are shown together with the corresponding ion score. The list of all the peptides identified for each spot is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone.0038356.s004" target="_blank">Table S1</a>.</p

Caspase activation after Epo removal.

<p>Western blotting (A) of total cell lysates from one independent culture, harvested after 6 hour, 12 hour and 24 hour in ESDL (+Epo) and SDL (-Epo) using an antibody against cleaved caspase 3, caspase 9, caspase 8, and hsc70 was used as a loading control. 20 µg of protein lysate was loaded in each well (B) Flow Cytometry analysis of active caspase 8, active caspase 3 and active caspase 9 for ESDL (thick black line) grown cells and SDL (dotted grey line) after 24 hours. Active caspase 9 was detected on a separate culture from the caspase 3 and caspase 9 and using a different flow cytometer.</p