[show abstract][hide abstract] ABSTRACT: An intriguing observation is that human erythrocytes can maintain their structural integrity over their 120-day life span despite the fact that they can no longer synthesize new proteins as they undergo enucleation upon maturation. Calmodulin (CaM), a canonical Ca 2+ binding protein, plays a key role in keeping homeostasis
[show abstract][hide abstract] ABSTRACT: Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca(2+) but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca(2+)-dependent manner. We have previously mapped a Ca(2+)-independent CaM-binding site, pep11 (A(264)KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca(2+) (Ca(2+)/CaM), interacts simultaneously with pep11 and with Ser(185) in A(181)KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca(2+)/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca(2+)/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca(2+). Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca(2+). Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca(2+)/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca(2+)/CaM-dependent regulation of R30 binding to membrane proteins.
Cell biochemistry and biophysics 10/2013; · 3.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120-day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor MetHb formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that MetHb binding to the cytoplasmic domain of band 3 in peroxidized, but not in untreated erythrocyte membranes, induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density-gradient procedure, have enabled us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ~ 0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced NADH-dependent reductase activity, and accumulated oxidative membrane damage. These findings have enabled us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably due to its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from the band 3, enabling the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.
[show abstract][hide abstract] ABSTRACT: The classical function of 4.1R in erythrocytes is to contribute to the mechanical properties of the membrane by promoting the interaction between spectrin and actin. It is now well recognized that 4.1R is required for the stable anchorage of numerous cell surface erythrocyte membrane proteins. 4.1R is the prototypical member of the family of 4.1 proteins which are expressed in many cell types besides erythrocytes. The other members of the protein 4.1 family include 4.1N, 4.1G and 4.1B. NHE1 (Na+/H+ exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes, supporting a functional interaction between NHE1 and 4.1R. We recently demonstrated that 4.1R binds directly to the cytoplasmic domain of NHE1 (NHE1cd). This interaction involves an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain and two clusters of basic amino acids in the NHE1cd, K519R and R556FNKKYVKK, previously shown to mediate PIP2 (phosphatidylinositol 4,5-bisphosphate) binding. The affinity of this interaction is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of electrostatic nature. The binding affinity is also reduced upon binding of Ca2+/CaM (Ca2+-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH as well as Na+ and Ca2+ concentrations. In this review, we discuss the increasing evidence for an important role for members of the protein 4.1 family of membrane skeletal proteins in the regulation of various ion transporters in erythrocytes and in non-erythroid cells.
[show abstract][hide abstract] ABSTRACT: Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca(2+)-saturated calmodulin (Ca(2+)/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin-radixin-moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca(2+)/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca(2+)/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca(2+)/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S(71)RGISRFIPPWLKKQKS peptide (pepG) mediates Ca(2+)/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca(2+)/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca(2+)/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.
Cell biochemistry and biophysics 01/2013; · 3.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: In human erythrocytes, the 80–kDa isoform of protein 4.1R, 4.1R80, maintains mechanical membrane stability and
deformability as a result of multiple protein–protein interactions. 4.1R80 binds to transmembrane proteins glycophorin C (GPC) and band 3 via its 30–kDa N–terminal FERM (Four one–Ezrin–Radixin–Moesin) domain (referred to as “R30” in the present study) and to spectrin and actin via a 10–kDa domain. Although the sites in R30 responsible for interaction with transmembrane proteins have been extensively studied, the identity of these sites has been challenged recently. Antibodies, in particular monoclonal antibodies (mAbs), are powerful tools not only for immunochemical studies but also for functional analyses such as the monitoring of the dynamic interactions of R30 with its binding partners. In the present study, we have generated mouse mAbs against recombinant R30 protein, and characterized their respective recognition epitopes in R30 using various recombinant proteins. Four representative clones, #5, #7, #9, #13 recognized the Y131DPELHGVDYVSDFKLAPN (pep8.1), Q150 TKELEEKVMELHKSYR (pep8.2), M1HCKVSLLDDTVYECVVE (pep4) and Q247EQYESTIGFKLPSYRA (pep13) epitopes, respectively. These sequences are located in the N–,α– and C–lobe structure of R30, respectively. IAsys–based in vitro binding analyses enabled us to demonstrate that the binding of R30 to pH 11–treated inside–out–vesicles (IOVs) was reduced by two combinations of mAbs (#5 and #9 or #7 and #9) but not by any of the mAbs alone and to confirm that the GPC binding site of R30 was located at or near pep8.1 and pep8.2 in the α– lobe of R30. Our study validates that this novel panel of mAbs constitutes a powerful tool for various types of analyses of 4.1R.
[show abstract][hide abstract] ABSTRACT: The membrane skeleton plays a central role in maintaining the elasticity and stability of the erythrocyte membrane, two biophysical features critical for optimal functioning and survival of red cells. Many constituent proteins of the membrane skeleton are phosphorylated by various kinases, and phosphorylation of β-spectrin by casein kinase and of protein 4.1R by PKC has been documented to modulate erythrocyte membrane mechanical stability. In this study, we show that activation of endogenous PKA by cAMP decreases membrane mechanical stability and that this effect is mediated primarily by phosphorylation of dematin. Co-sedimentation assay showed that dematin facilitated interaction between spectrin and F-actin, and phosphorylation of dematin by PKA markedly diminished this activity. Quartz crystal microbalance measurement revealed that purified dematin specifically bound the tail region of the spectrin dimer in a saturable manner with a submicromolar affinity. Pulldown assay using recombinant spectrin fragments showed that dematin, but not phospho-dematin, bound to the tail region of the spectrin dimer. These findings imply that dematin contributes to the maintenance of erythrocyte membrane mechanical stability by facilitating spectrin-actin interaction and that phosphorylation of dematin by PKA can modulate these effects. In this study, we have uncovered a novel functional role for dematin in regulating erythrocyte membrane function.
Journal of Biological Chemistry 08/2012; 287(42):35244-50. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations.
[show abstract][hide abstract] ABSTRACT: Although the 3D structure of the Ca(2+)-bound CaM (Ca(2+)/CaM) complex with the antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), has been resolved, the dynamic changes in Ca(2+)/CaM structure upon interaction with W-7 are still unknown. We investigated time- and temperature-dependent dynamic changes in Ca(2+)/CaM interaction with W-7 in physiological conditions using one- and two-dimensional Fourier-transformed infrared spectroscopy (2D-IR). We observed changes in the α-helix secondary structure of Ca(2+)/CaM when complexed with W-7 at a molar ratio of 1:2, but not at higher molar ratios (between 1:2 and 1:5). Kinetic studies revealed that, during the initial 125s at 25°C, Ca(2+)/CaM underwent formation of secondary coil and turn structures upon binding to W-7. Variations in temperature that induced significant changes in the structure of the Ca(2+)/CaM complex failed to do so when Ca(2+)/CaM was complexed with W-7. We concluded that W-7 induced stepwise conformational changes in Ca(2+)/CaM that resulted in a rigidification of the complex and its inability to interact with target proteins and/or polypeptides.
Biochemical and Biophysical Research Communications 06/2012; 423(2):360-5. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interaction of protein 4.1 (4.1R) with the transmembrane protein glycophorin C (GPC) regulates the functions of erythrocyte membrane. Fluorescence correlation spectroscopy (FCS) was used to define the interaction of EGFP-4.1R with DsRed-GPC on transport vesicles (TVs) by measuring their fluctuation in living cells. DsRed-GPC expressed in HeLa cells was delivered to the plasma membrane through slow vesicle transport. EGFP-4.1R, which freely diffused in the cytosol when expressed alone, diffused slowly when co-expressed with DsRed-GPC, indicating association of EGFP-4.1R with TVs. Fluorescence cross-correlation spectroscopy (FCCS) showed direct interaction of EGFP-4.1R with DsRed-GPC on TVs. The present study demonstrates that 4.1R binds to GPC on TVs in living cells.
[show abstract][hide abstract] ABSTRACT: In this study, we describe a novel application for light scattering, a method widely used for separation of molecules in solution based on their size. We demonstrate that light scattering analysis can monitor the change in particle size of protein 4.1R prior to and after binding to red blood cell inside-out-vesicles in solution. Light scattering constitutes therefore a novel tool to analyze protein-binding association constants.
[show abstract][hide abstract] ABSTRACT: Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.
[show abstract][hide abstract] ABSTRACT: In erythrocytes, 4.1R80 (80 kDa isoform of protein 4.1R) binds to the cytoplasmic tail of the transmembrane proteins band 3 and GPC (glycophorin C), and to the membrane-associated protein p55 through the N- (N-terminal), α- (α-helix-rich) and C- (C-terminal) lobes of R30 [N-terminal 30 kDa FERM (4.1/ezrin/radixin/moesin) domain of protein 4.1R] respectively. We have shown previously that R30 binds to CaM (calmodulin) in a Ca2+-independent manner, the equilibrium dissociation constant (Kd) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30's structural stability using resonant mirror detection and FTIR (Fourier-transform IR) spectroscopy. After a 30 min incubation above 40° C, R30 could no longer bind to band 3 or to GPC. In contrast, R30 binding to p55, which could be detected at a temperature as low as 34° C, was maintained up to 44° C in the presence of apo-CaM. Dynamic light scattering measurements indicated that R30, either alone or complexed with apo-CaM, did not aggregate up to 40° C. FTIR spectroscopy revealed that the dramatic variations in the structure of the β-sheet structure of R30 observed at various temperatures were minimized in the presence of apo-CaM. On the basis of Kd values calculated at various temperatures, ΔCp and ΔG° for R30 binding to apo-CaM were determined as -10 kJ · K(-1) · mol-1 and ~ -38 kJ · mol(-1) at 37° C (310.15 K) respectively. These data support the notion that apo-CaM stabilizes R30 through interaction with its β-strand-rich C-lobe and provide a novel function for CaM, i.e. structural stabilization of 4.1R80.
[show abstract][hide abstract] ABSTRACT: The human ether-a-go-go-related gene (hERG) protein is a cardiac potassium channel. Mutations in hERG can result in reductions in membrane channel current, cardiac repolarization, prolongation of QT intervals, and lethal arrhythmia. In the last decade, it has been found that some mutants of hERG involved in long QT syndrome exhibit intracellular protein trafficking defects, while other mutants sort to the membrane but cannot form functional channels. Due to the close relationship between intracellular trafficking and functional protein expression, we aimed to measure differences in protein behavior/motion between wild-type and mutant hERG by directly analyzing the fluorescence fluctuations of green fluorescent protein-labeled proteins using fluorescence correlation spectroscopy (FCS). Our data imply that FCS can be applied as a new diagnostic tool to assess whether the defect in a particular mutant channel protein involves aberrant intracellular trafficking.
The Journal of Physiological Sciences 05/2011; 61(4):313-9. · 1.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R(135) and 4.1R(80)), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R(80) is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R(80) and 4.1R(135) to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R(135). We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains.
International Journal of Cell Biology 01/2011; 2011:943272.
[show abstract][hide abstract] ABSTRACT: Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. Although the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high-affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.
[show abstract][hide abstract] ABSTRACT: Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.
Journal of Biological Chemistry 10/2010; 285(44):33923-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vβ3(+)CD4(+) T cells exhibited a high level of expansion whereas the Vβ11(+)CD4(+) T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vβ11(+)CD4(+) T cells exhibited a high level of expansion while the Vβ3(+)CD4(+) T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vβ molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vβ3(+)CD4(+) and Vβ11(+)CD4(+) T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vβ3/Vβ11 β chains and between SEA-MHC class II-molecule complex and Vβ3(+)/Vβ11(+) CD4(+) T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.
Journal of Biological Chemistry 10/2010; 285(40):30427-35. · 4.65 Impact Factor