Human Erythrocyte Protein Research Articles

Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex

GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l(-1)·min(-1), respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex.

Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

Critical band 3 multiprotein complex interactions establish early during human erythropoiesis

AbstractBand 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.

Comprehensive Identification of Glycated Peptides and Their Glycation Motifs in Plasma and Erythrocytes of Control and Diabetic Subjects

Nonenzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.

ChemInform Abstract: Three‐Dimensional Structure of Membrane Protein Stomatin and Function of Stomatin‐Specific Protease

AbstractReview: 18 refs.

Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes

Noncovalent DIDS binding to Band 3 (AE1) protein in human erythrocyte membranes, modified by non-penetrating, water soluble 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC), was studied at 0°C in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ± 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for “DIDS-Band 3” complex, determined from the chloride transport experiments, was (34 ± 3) nM and (80 ± 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ± 6) nM and (60 ± 8) nM in control and EAC-treated ghosts, respectively. The reduced affinity for DIDS reversible binding was not a result of negative cooperativity of DIDS binding sites of Band 3 oligomer since Hill’s coefficients were indistinguishable from 1 (within the limit error) both for control and EAC-treated ghosts. By using tritium-labeled DIDS, 4,4’-diisothiocyanato-2,2’-stilbenedisulfonate ([3H]DIDS), the association rate constant, k+1 (M−1s−1), was measured. The mean values of (4.3 ± 0.7) × 105 M−1s−1 for control and (2.7 ± 0.7) × 105 M−1s−1 for EAC-treated ghosts were obtained. The mean values for KD, evaluated from [3H]DIDS binding measurements, were (37 ± 9) nM and (90 ± 21) nM for control and EAC-modified ghosts, respectively. The results demonstrate that EAC modification of AE1 reduces about 2-fold the affinity of AE1 for DIDS. It is suggested that half of the subunits are modified near the transport site by EAC.

Three-dimensional Structure of Membrane Protein Stomatin and Function of Stomatin-specific Protease

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. It is reported that stomatin regulates the gating of acid-sensing ion channels in mammalian neurons. However, the function of stomatin is not fully understood. In the genomic sequence of the hyperthermophilic archaeon Pyrococcus horikoshii, the putative operon-forming genes PH1511 and PH1510 encode stomatin and its partner protein, respectively. The N-terminal region of PH1510p (1510-N) is a serine protease, and specifically cleaves the C-terminal hydrophobic region of stomatin PH1511p. We have determined the first crystal structure of the core domain of stomatin PH1511p (residues 56-234, designated as PhSto(CD)). This review focuses on the three-dimensional structure of PhSto(CD), and discusses the function of stomatin and its specific protease 1510-N. PhSto(CD) forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto(CD) is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows a conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled-coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. The coiled-coil fold observed in the crystal probably contributes to the self-association.

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.

Cluster Analysis for the Impact of Sickle Cell Disease on the Human Erythrocyte Protein Interactome

Protein interaction networks provide useful information to assess impacts of disease on cell functions. Statistical clustering methods applied to these networks can reveal impacts on particular functional groups of proteins. In addition, clustering methods can identify subsets of proteins that require additional study to provide updated information regarding their position within an interaction network, and hence increase our understanding of their relationships with other proteins in the network. These ideas are illustrated here by considering the impacts of sickle cell disease on the human erythrocyte interaction network. Statistical cluster analyses are performed based on a measure of similarity for nodes within a network called the Generalized Topological Overlap Measure. These analyses identify clusters of proteins that are similar in terms of shared interaction partners. Identification of clusters that contain proteins whose relative abundances have been significantly altered in sickle cell patients provides specific information about the impact of these proteins on erythrocyte functions.

Transmembrane Helix Association Affinity Can Be Modulated by Flanking and Noninterfacial Residues

The GxxxG sequence motif mediates the association of transmembrane (TM) helices by providing a site of close contact between them. However, it is not sufficient for strong association. For example, both bacteriophage M13 major coat protein (MCP) and human erythrocyte protein glycophorin A (GpA) contain a GxxxG motif in their TM domains and form a homodimer, but the association affinity of MCP, measured by the ToxCAT in vivo assay, is dramatically weaker than that of GpA. Even when all interfacial residues of MCP were substituted for those of GpA (MCP-GpA), association remained significantly weaker than in GpA. Here we provide an explanation for these experimental observations using molecular dynamics simulations in an implicit membrane (IMM1-GC). The association free energies of GpA29 (GpA with 29 residues all from the wild-type sequence), GpA15p11 (GpA with 15 residues from the wild-type sequence plus 11 flanking residues from the ToxCAT construct), MCP, and MCP-GpA TM helices were calculated and compared. MCP and MCP-GpA have the same flanking residues used in the ToxCAT assay as those in GpA15p11, but the position of the flanking residues relative to the GxxxG motif is different. The calculated association free energies follow experimental observations: the association affinity of MCP-GpA falls between those of GpA15p11 and MCP wild-type. MCP exhibits an equally strong interhelical interaction in the TM domain. A major reason for the weaker association of MCP in the calculations was the noninterfacial residue Lys-40, which in the dimer structure is forced to be buried in the membrane interior. To alleviate the desolvation cost, in MCP and MCP-GpA dimers, Lys-40 gets deprotonated. A second factor that modulates association affinity is the flanking residues. Thanks to them, GpA15p11 exhibits a much stronger association affinity than GpA29. The positioning of the flanking residues is also important, as evidenced by the difference in association affinity between MCP and MCP-GpA on one hand and GpA15p11 on the other. Thus, residues outside the contact interface can exert a significant influence on transmembrane helix association affinity.

Site-specific GlcNAcylation of human erythrocyte proteins: potential biomarker(s) for diabetes.

OBJECTIVE—O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals.RESEARCH DESIGN AND METHODS—GlcNAcylated erythrocyte proteins or GlcNAcylated peptides were tagged and selectively enriched by a chemoenzymatic approach and identified by mass spectrometry. The enrichment approach was combined with solid-phase chemical derivatization and isotopic labeling to detect O-GlcNAc modification sites and to compare site-specific O-GlcNAc occupancy levels between normal and diabetic erythrocyte proteins.RESULTS—The enzymes that catalyze the cycling (addition and removal) of O-GlcNAc were detected in human erythrocytes. Twenty-five GlcNAcylated erythrocyte proteins were identified. Protein expression levels were compared between diabetic and normal erythrocytes. Thirty-five O-GlcNAc sites were reproducibly identified, and their site-specific O-GlcNAc occupancy ratios were calculated.CONCLUSIONS—GlcNAcylation is differentially regulated at individual sites on erythrocyte proteins in response to glycemic status. These data suggest not only that site-specific O-GlcNAc levels reflect the glycemic status of an individual but also that O-GlcNAc site occupancy on erythrocyte proteins may be eventually useful as a diagnostic tool for the early detection of diabetes.

Dematin and Adducin Provide a Novel Link between the Spectrin Cytoskeleton and Human Erythrocyte Membrane by Directly Interacting with Glucose Transporter-1

Dematin and adducin are actin-binding proteins located at the spectrin-actin junctions, also called the junctional complex, in the erythrocyte membrane. Here we propose a new model whereby dematin and adducin link the junctional complex to human erythrocyte plasma membrane. Using a combination of surface labeling, immunoprecipitation, and vesicle proteomics approaches, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the junctional complex to the lipid bilayer. Because homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

Crystal Structure of a Core Domain of Stomatin from Pyrococcus horikoshii Illustrates a Novel Trimeric and Coiled-Coil Fold

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56–234 of PH1511p, designated as PhSto CD). PhSto CD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments of about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto CD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto CD would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.

The 1.3-A resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins.

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels. We have now solved the structure of a rare bacterial homologue (from Nitrosomonas europaea) of human Rh50 proteins at a resolution of 1.3 A. The protein is a trimer, and analysis of its subunit interface strongly argues that all Rh proteins are likely to be homotrimers and that the human erythrocyte proteins RhAG and RhCE/D are unlikely to form heterooligomers as previously proposed. When compared with structures of bacterial Amt proteins, NeRh50 shows several distinctive features of the substrate conduction pathway that support the concept that Rh proteins have much lower ammonium affinities than Amt proteins and might potentially function bidirectionally.

Exercise training in normobaric hypoxia: is carbonic anhydrase III the best marker of hypoxia?

LETTERS TO THE EDITORExercise training in normobaric hypoxia: is carbonic anhydrase III the best marker of hypoxia?Jaume Padilla, Sarah A. Hamilton, Elizabeth A. Lundgren, James M. McKenzie, and Timothy D. MickleboroughJaume Padilla, Sarah A. Hamilton, Elizabeth A. Lundgren, James M. McKenzie, and Timothy D. MickleboroughPublished Online:01 Aug 2007https://doi.org/10.1152/japplphysiol.00408.2007MoreSectionsPDF (28 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat To the Editor: We read with great interest a series of three articles (1, 4, 5) published in the April 2006 issue of the Journal of Applied Physiology examining the effect of exercise training in normobaric hypoxia on aerobic performance capacity, muscular adjustments of selected gene transcripts, and skeletal muscle mitochondrial properties in endurance runners. The authors found an improvement in V̇o2max, an increase in time to exhaustion (Tlim) at the velocity at V̇o2max, simultaneous mitochondrial adaptations, and increases in transcriptional factors implicated in skeletal muscle adaptation. These findings have substantial implications for athletes undergoing endurance training programs in hypoxia. However, as we critically reviewed the papers, we found several points that require clarification. First, although we agree that carbonic anhydrase (CA) was significantly upregulated after training in hypoxia, there is a lack of rationalization for the use of the CA3 gene as a marker of hypoxia. Other isoenzymes of CA (CA4 and -14) have been shown to be upregulated in response to chronic hypoxia (2) but it is questionable whether or not this is true for CA3. Juel et al. (2) found no significant changes in CA3 in response to hypoxia. In addition, a murine study by Kim et al. (3) investigated the significance of this gene and found that it was not affected by changes in environmental oxygen alterations. Could the authors discuss their logic for choosing CA3 instead of a more established isoenzyme of CA that marks hypoxia? Second, inconsistent sample size across manuscripts, without adequate justification, was observed. Third, in all three articles (1, 4, 5), the authors used a two-way ANOVA with one repeated factor, which is the appropriate model for their statistical analysis. However, in the results, they do not report the group by time interaction although this interaction is necessary to justify a statistically different response to endurance training between groups. Fourth, Fig. 2 of the third article (5) illustrates the correlation between mRNA levels and Tlim combining pre- and post-training values. It is assumed that data were presented in absolute terms, however, the range of values for Tlim (0–20 s) do not make sense as presented.REFERENCES1 Dufour SP, Ponsot E, Zoll J, Doutreleau S, Lonsdorfer-Wolf E, Geny B, Lampert E, Fluck M, Hoppeler H, Billat V, Mettauer B, Richard R, Lonsdorfer J. Exercise training in normobaric hypoxia in endurance runners. I. Improvement in aerobic performance capacity. J Appl Physiol 100: 1238–1248, 2006.Link | ISI | Google Scholar2 Juel C, Lundby C, Sander M, Calbet JAL, Van Hall G. Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia. J Physiol 548: 639–648, 2003.Crossref | PubMed | ISI | Google Scholar3 Kim G, Lee T, Wetzel P, Geers C, Robinson MA, Myers TG, Owens JW, Wehr NB, Eckhaus MW, Gros G, Wynshaw-Boris A, Levine RL. Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span. Mol Cell Biol 24: 9942–9947, 2004.Crossref | PubMed | ISI | Google Scholar4 Ponsot E, Dufour SP, Zoll J, Doutreleau S, N'Guessan B, Geny B, Hoppeler H, Lampert E, Mettauer B, Ventura-Clapier R, Richard R. Exercise training in normobaric hypoxia in endurance runners. J Appl Physiol 100: 1249–1257, 2006.Link | ISI | Google Scholar5 Zoll J, Ponsot E, Dufour SP, Doutreleau S, Ventura-Clapier R, Vogt M, Hoppeler H, Richard R, Fluck M. Exercise training in normobaric hypoxia in endurance runners. III. Muscular adjustments of selected gene transcripts. J Appl Physiol 100: 1258–1266, 2006.Link | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: J. Padilla, Indiana Univ. (HPER 112), Dept. of Kinesiology, Bloomington, IN 47405 (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationCited ByNoninvasive 64 Cu-ATSM and PET/CT Assessment of Hypoxia in Rat Skeletal Muscles and Tendons During Muscle Contractions14 May 2009 | Journal of Nuclear Medicine, Vol. 50, No. 6Reply to Padilla, Hamilton, Lundgren, Mckenzie, and MickleboroughJoffrey Zoll, Elodie Ponsot, Stephane Dufour, and Martin Flück1 August 2007 | Journal of Applied Physiology, Vol. 103, No. 2 More from this issue > Volume 103Issue 2August 2007Pages 730-730 Copyright & PermissionsCopyright © 2007 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00408.2007PubMed17666736History Published online 1 August 2007 Published in print 1 August 2007 Metrics

Comparison of the effect of phenol and its derivatives on protein and free radical formation in human erythrocytes ( in vitro)

The effect of phenolic compounds: phenol, 2,4–dichlorophenol (2,4-DCP), 2,4-dimethylphenol (2,4-DMP) and catechol on human erythrocytes was studied. The level of fluorescent label – 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H 2DCFDA) oxidation by phenolic compounds in erythrocytes as well as the carbonyl group content and hemoglobin denaturation were monitored. H 2DCFDA has been utilized extensively as a marker for studies of oxidative stress at the cellular level. We noted that 2,4-DCP, 2,4-DMP and catechol induced an increase in the concentration- and time-dependent H 2DCFDA oxidation. We also observed an increase in carbonyl group content and the changes in parameter T (denaturation of hemoglobin) in erythrocytes incubated with 2,4-DCP, catechol and 2,4-DMP. The highest level of H 2DCFDA oxidation was provoked by 2,4-DCP. The biggest changes of proteins in erythrocytes measured as the carbonyl group content were induced by 2,4-DMP, but measured as parameter T they were induced by catechol. It was observed that phenol did not oxidize H 2DCFDA up to the concentration of 2.5 mM after 3 h of incubation. Phenol did not affect the carbonyl group content but decreased parameter T (induced denaturation of hemoglobin). To sum up, the kind of the substituent in a phenolic ring determines the molecular mechanism of action of the individual compound and the capacity of reactive oxygen species generation and thus damages the specified structures in human erythrocytes.

Tropomyosin modulates erythrocyte membrane stability

AbstractThe ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a β-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex.

Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer.

The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG(79)VMAGVIGTILLISWG(94)IKK was labeled at G(79) and G(94) with (13)C=(16)O or (13)C=(18)O. The isotopomers and their 50:50 mixtures formed helical dimers in SDS micelles whose 2D IR spectra showed components from homodimers when both helices had either (13)C=(16)O or (13)C=(18)O substitution and a heterodimer when one had (13)C=(16)O substitution and the other had (13)C=(18)O substitution. The cross-peaks in the pure heterodimer 2D IR difference spectrum and the splitting of the homodimer peaks in the linear IR spectrum show that the amide-I mode is delocalized across a pair of helices. The excitation exchange coupling in the range 4.3-6.3 cm(-1) arises from through-space interactions between amide units on different helices. The angle between the two Gly(79) amide-I transition dipoles, estimated at 103 degrees from linear IR spectroscopy and 110 degrees from 2D IR spectroscopy, combined with the coupling led to a structural picture of the hydrophobic interface that is remarkably consistent with results from NMR on helix dimers. The helix crossing angle in SDS is estimated at 45 degrees. Two-dimensional IR spectroscopy also sets limits on the range of geometrical parameters for the helix dimers from an analysis of the coupling constant distribution.

Phosphatidylinositol-4,5-Biphosphate (PIP2) Differentially Regulates the Interaction of Human Erythrocyte Protein 4.1 (4.1R) with Membrane Proteins

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types.

Two Distinct Domains Mediate Self-Assembly of Knob Protein in Plasmodium Falciparum-Infected Human Erythrocytes.

Plasmodium falciparum associated mortality is believed to originate from the adhesion of infected erythrocytes to the endothelial cells. The adhesion of mature parasite infected erythrocytes is mediated by the surface protrusions called knobs. A dominant constituent of knobs is the parasite derived knob-associated histidine-rich protein termed KAHRP or knob protein. Gene deletion experiments have shown that the knob protein is essential in malaria pathogenesis. Without the knob protein (KAHRP), adherence of malaria-infected erythrocytes is not established under physiological shear stress. The adhesive properties of infected erythrocytes are propagated by the direct interaction of KAHRP, a peripheral membrane protein, with the cytoplasmic domain of the P. falciparum erythrocyte membrane protein-1 (PfEMP1). Unlike the highly polymorphic extracellular domain, the cytoplasmic domain of PfEMP1 is conserved across species, hence the interest in delineating the mechanism of its interaction with KAHRP and components of the erythrocyte membrane. Our studies have observed recombinant KAHRP to self-associate into large aggregates in vitro. Under transmission electron microscopy, both knobs isolated from malaria-infected erythrocytes and self-assembled recombinant KAHRP in vitro have revealed a unique spiral shaped morphology. Based on these observations, we hypothesized that the unique knob spiral is formed by the self-assembly of KAHRP monomers and therefore must be encoded by distinct sequence elements within KAHRP. To investigate this speculation, we devised a solution binding assay and engineered a series of GST (glutathione S-transferase) and Trx (thioredoxin) fusion peptides to encompass the entire length of KAHRP. Binding measurements using heterologous combination of defined fusion peptides reveal a core histidine-rich amino terminal segment associating with a lysine-rich segment located within the carboxyl half of KAHRP. These findings suggest a novel head-to-tail interaction strategy employed by the intracellular parasite to assemble the spiral-shaped scaffold of KAHRP under the surface of electron dense knobs. Significantly, our results identify a novel self-interaction site within KAHRP that could be amenable to therapeutic intervention against cerebral malaria.