Synthesis and expression of cell-surface glycoproteins during chick erythroid differentiation

Abstract. A panel of monoclonal antibodies to differentiation antigens on avian erythroid cells was used to study the reprogramming of protein synthesis during erythroid differentiation at the molecular level. This panel detected five distinct cell-surface glycoproteins on immature leukemic erythroblasts, all of which were initially synthesised as smaller intracellular precursors. Two distinct in vitro differentiation systems (erythroblasts transformed by ts mutants of the erb-B and sea retroviral oncogenes, in which the synchronous terminal differentiation of CFU-E-like precursors is induced by simple elevation of temperature) were used to study cell-surface expression and the biosynthesis of each protein during erythroid cell maturation. For four glycoproteins, both cell-surface expression and biosynthesis decreased between the erythroblast and erythrocyte stages, although with widely different time courses. The fifth glycoprotein, which is reticulocyte specific on normal erythroid progenitors and is aberrantly expressed in onco-gene-transformed erythroblasts, rapidly disappeared shortly after differentiation induction but was then re-expressed on reticulocytes with the same time course as that seen during normal erythroid differentiation. This indicates that ts erb-B- and ts sea -transformed erythroblasts revert to a normal precursor phenotype before undergoing temperature-induced differentiation.     

Platelet adhesion to collagen. Factors affecting Mg2(+)-dependent and bivalent-cation-independent adhesion.

Platelet adhesion to collagens immobilized on plastic has been measured, with the following results. (1) Human, but not rabbit, platelets adhered readily to pepsin-extracted monomeric collagens in an Mg2(+)-dependent manner. (2) Rabbit platelets adhered to a monomeric collagen extracted without pepsin by a process that was cation-independent; human platelet adhesion to this collagen exhibited a cation-independent element. (3) Human platelet adhesion to polymeric collagens, including intact native fibres and those reconstituted from pepsin-extracted monomeric collagens, exhibited appreciable cation-independence; adhesion of rabbit platelets to these collagens occurred only by a cation-independent process; pepsin treatment of the intact fibres caused a reduction in cation-independent binding. Two mechanisms of adhesion can therefore be distinguished, one Mg2(+)-dependent, expressed by human, but not rabbit, platelets, the other cation-independent and exhibited by platelets of both species. Mg2(+)-dependent and cation-independent adhesion sites are located within the triple helix of collagen, but the latter sites are only expressed in collagen in polymeric form. In neither case is the helical conformation of the sites essential for their binding activity. Cation-independent adhesion sites are also located in the pepsin-sensitive non-helical telopeptides of collagen and can be expressed in both monomeric and polymeric collagens. Chemical modification of collagen lysine residues indicates that specific lysine residues may be involved in Mg2(+)-dependent adhesion. Adhesion using human citrated platelet-rich plasma is Mg2(+)-independent. Plasma contains factors, conceivably the adhesive proteins fibronectin and von Willebrand factor, that promote the Mg2(+)-independent mechanism.     

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation.     

Monomeric Creatine Kinase Aggregation and Sodium Dodecyl Sulfate-cyclodextrin Assisted Refolding

Abstract

The monomelic state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a dénaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as α-, β-, and γ-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS- binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.     

Hemoglobin switching in sheep: commitment of erythroid stem cells to expression of the betaC-globin gene and accumulation of betaC-globin mRNA

The switch from HbA (α₂β₂^A) to HbC (α₂β₂^C) synthesis was induced by injection of erythropoietin into a lamb homozygous for HbA. Serial samples of bone marrow were analyzed to detect the initial commitment of erythroid stem cells (CFU-E) to form colonies which made HbC in vitro, and to detect the initial accumulation of β^C-globin mRNA and the onset of HbC synthesis in erythroblasts in vivo. CFU-E-derived erythroid colonies were formed in plasma clot culture at a low erythropoietin concentration, and the relative amounts of β^A- and β^C-globin synthesized were determined after a 24 hr pulse of ³H-leucine, added after 84 hr in culture. RNA was extracted from nuclei and cytoplasm of “early” and “late” populations of bone marrow erythroblasts which had been fractionated by Ficoll-Hypaque density centrifugation. The concentration of β^A- and β^C-globin mRNA was determined by annealing to purified synthetic DNAs (cDNAs) complementary to β^A and β^C mRNA. No β^C-globin was synthesized in erythroblasts or in CFU-E-derived erythroid colonies prior to the injection of erythropoietin. An increase in the concentration of CFU-E in the bone marrow and the appearance of β^C-globin synthesis in CFU-E-derived colonies were detected 12 hr after the erythropoietin injection. In contrast, β^C mRNA was not detected in either “early” or “late” erythroid cells until 36 hr later. The first measurable β^C-globin mRNA was accompanied by the appearance of β^C-globin synthesis in bone marrow erythroblasts. Our results suggest that the accumulation of β^C-globin mRNA is a relatively late event following induction of HbA to HbC switching by erythropoietin. The expansion of the compartment of erythroid stem cells and the commitment of CFU-E to β^C-globin synthesis appear to precede the detectable accumulation of β^C mRNA by 24–36 hr.     

v-erbA cooperates with sarcoma oncogenes in leukemic cell transformation

The v-erbB, v-src, v-fps, v-sea, and v-Ha-ras oncogenes induce avian erythroid progenitor cells to self-renew in an erythropoietin-independent manner. These transformed erythroblasts retain both their capacity to differentiate into erythrocytes and their requirement for complex growth media. However, previous studies showed that erythroblasts transformed by v-erbB plus v-erbA (which by itself is not oncogenic) are blocked in differentiation and grow in standard media. Here we show that the introduction of v-erbA into erythroblasts transformed with v-src, v-fps, v-sea, or v-Ha-ras likewise induces a fully transformed phenotype. It also reduces the capacity of ts sea- and ts erbB-transformed erythroblasts to differentiate terminally in an erythropoietin-dependent manner after a temperature shift. Cooperativity involving v-erbA also occurs in vivo since chicks infected with a retroviral construct encoding v-erbA and v-src develop both acute erythroblastosis and sarcomas.     

Platelet-reactive sites in collagens type I and type III. Evidence for separate adhesion and aggregatory sites.

The adhesion of human and rabbit platelets to collagens and collagen-derived fragments immobilized on plastic was investigated. Adhesion appeared to be independent of collagen conformation, since similar attachment occurred to collagen (type I) in monomeric form, as fibres or in denatured state. The adhesion of human platelets was stimulated to a variable degree by Mg2+, but rabbit platelet adhesion showed little if any dependence on this cation. Collagens type I, III, V and VI were all able to support adhesion, although that to collagen type V (native) was lower than that to the other collagens. Adhesion to a series of peptides derived from collagens I and III was measured. Attachment did not require the presence of peptides in triple-helical configuration. The extent of adhesion ranged from relatively high, as good as to the intact parent collagen molecule, to little if any adhesive activity beyond the non-specific (background) level. The existence of very different degrees of activity suggests that platelet adhesion is associated with specific structural sites in the collagen molecule. Adhesion in many instances was essentially in accord with the known platelet-aggregatory activity of individual peptides. However, two peptides, alpha 1(I)CB3 and alpha 1(III)CB1,8,10,2, exhibited good adhesive activity although possessing little if any aggregatory activity. Of particular interest, despite its near-total lack of aggregatory activity, adhesion to peptide alpha 1(I)CB3 was as good as that to the structurally homologous peptide alpha 1(III)CB4, in which is located a highly reactive aggregatory site. This implies that platelet adhesion to collagen may involve sites in the collagen molecule distinct from those more directly associated with aggregation.     

The heavy-chain stoichiometry of smooth muscle myosin is a characteristic of smooth muscle tissues

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.     

Dissection of Conformational Conversion Events during Prion Amyloid Fibril Formation Using Hydrogen Exchange and Mass Spectrometry

A molecular understanding of prion diseases requires an understanding of the mechanism of amyloid fibril formation by the prion protein. In particular, it is necessary to define the sequence of the structural events describing the conformational conversion of monomeric PrP to aggregated PrP. In this study, the sequence of the structural events in the case of amyloid fibril formation by recombinant mouse prion protein at pH 7 has been characterized by hydrogen–deuterium exchange and mass spectrometry. The observation that fibrils are substantially more stable to hydrogen–deuterium exchange than is native monomer allows both forms to be quantified during the course of the aggregation reaction. Under the aggregation conditions utilized, native monomeric protein and amyloid fibrils are the only forms of the protein detectable during the course of the fibril formation reaction, suggesting that monomer directly adds on to the fibril template. Conformational conversion is shown to occur in two steps after the binding of monomer to fibril, with helix 1 unfolding only after helices 2 and 3 transform into β-sheet. Local stability in the β-sheet core region (residues ~ 159–225) of the fibrils is shown to be sequence dependent in that it varies along the length of the core, and local stability in protein molecules that are ordered in the structurally heterogeneous sequence segment 109–132 is shown to be similar to that in the core. This new understanding of the structural events during prion protein aggregation has important bearing on our comprehension of the molecular basis of prion pathogenesis.     

Monomeric creatine kinase aggregation and sodium dodecyl sulfate-cyclodextrin assisted refolding

The monomeric state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a denaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as alpha-, beta-, and gamma-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS-binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.     

Maturation of murine erythroleukemia cells committed to differentiation requires protein kinase C.

Treatment of murine erythroleukemia cells (MELC) attached to fibronectin-coated dishes with dimethyl sulfoxide causes the cells to become committed to the erythroid differentiation pathway. These cells mature extensively and acquire the characteristics of erythroid cells. The cells lose their cell-surface fibronectin receptors and accumulate red cell-specific membrane proteins, such as band 3, in amounts comparable to those in erythrocytes. Previous studies of MELC have shown that the presence of protein kinase C (PKC) is required for commitment to differentiation, but that the level of PKC activity declines progressively during maturation. In this study, we have established a role for PKC in the maturation of MELC committed to differentiation. Our results show that down-regulation of PKC by addition of phorbol 12-myristate 13-acetate (PMA) to committed MELC blocks subsequent maturation of the cells. Treatment of MELC with the PKC inhibitors H7 and sphingosine had similar effects. Down-regulation of PKC was assayed by measuring cytosolic PKC activity as well as by Western blotting using PKC antibodies. MELC maturation was monitored by loss of the cell-surface fibronectin receptor, release of cells from fibronectin plates, and accumulation of the band 3 anion transport protein. Immunoprecipitation of surface-labeled proteins by an anti-fibronectin receptor (integrin) antibody showed that PMA-treated cultures had more fibronectin receptor protein than untreated cultures 6 days post-induction. As a result, cultures of committed MELC treated with PMA remained attached to fibronectin-coated plates, whereas non-PMA-treated cells were released into the culture medium. Furthermore, PKC-depleted cells accumulated much smaller amounts of band 3 protein and band 3 mRNA than did non-PKC-depleted controls. Our results show that although PKC activity declines progressively during post-commitment maturation of MELC, its continued presence is critical for the process of cellular maturation.     

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells

Binding experiments with the specific muscarinic ligand [³H]N-methylscopolamine (³H-NMS) have shown the presence of constitutive muscarinic acetylcho-line receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1–M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K_D) for ³H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B⁺) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B⁺ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells. J. Cell. Physiol. 178:333–340, 1999. © 1999 Wiley-Liss, Inc.     

Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC₅₀ of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.     

Beta-amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH.

Amyloid protein (Abeta1-40) aggregation and conformation was examined using native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the results compared with those obtained by atomic force microscopy, and with Congo red binding, sedimentation and turbidity assays. The amount of Abeta aggregation measured was different, depending upon the method used. Incubation for 15 min at pH 5.0 or in the presence of Fe2+, Cu2+ or Zn2+ did not alter the level of Abeta oligomers observed on SDS and native gels. However, the slow aggregation of Abeta to form high molecular mass species over 5 days was inhibited. In contrast, when Abeta aggregation was monitored using a Congo red binding assay or sedimentation assay, a rapid increase in Abeta aggregation was observed after incubation for 15 min at pH 5.0, or in the presence of Fe2+, Cu2+ or Zn2+. The low pH-, Zn2+- or Cu2+-induced Abeta aggregation measured in a turbidity assay was reversible. In contrast, a considerable proportion of the Abeta aggregation measured by native and SDS/PAGE was stable. Atomic force microscopy studies showed that Abeta aged at pH 5.0 or in the presence of Zn2+ produced larger looser rod-shaped aggregates than at pH 7.4. Abeta that had been aged at pH 7.4 was more cytotoxic than Abeta aged at pH 5.0. Taken together, the results suggest that Abeta oligomerizes via two mutually exclusive mechanisms to form two different types of aggregates, which differ in their cytotoxic properties.