Regulation of erythrocyte membrane protein gene expression

Proteins of the erythrocyte membrane have served as the prototypes of homologous families of multifunctional proteins in erythroid and nonerythroid cells. These proteins demonstrate many different cell type, tissue-specific, and developmental stage-specific functions. This complex pattern of functional diversity appears to have evolved from the cell type, tissue-specific, developmentally regulated expression of multiple protein isoforms. Isoform diversity arises from different gene products from related genes; from differential, alternate splicing of the same gene product; from the use of tissue-specific promoters; and from alternate polyadenylation. The identification and characterization of the regulatory elements that control erythrocyte membrane protein gene expression have important implications for several biologic processes. These include disease pathogenesis, membrane assembly, hematopoiesis, gene regulation, and direction of other erythroid-specific genes in transgenic mouse and gene therapy applications.     

Effect of complement on the viscoelastic properties of human erythrocyte membrane

Using the micropipette technique, we examined the viscoelastic properties of the red blood cell (RBC) membrane which had been strongly coated with various components of complement (primary C3b, C3d, C4b, C4d) in vitro. The membrane elastic modulus (E), the viscosity index of the initial rapid phase of deformation (eta D1), the viscosity of the later slow phase of deformation (eta D2) and the viscosity of the recovery phase (eta R) were determined. Compared to control non-complement coated RBCs, RBCs coated with C3d, either alone or with other complement components, showed significant increase in the values for elasticity and viscosities. Thus C3d fixation resulted in decreased membrane deformability. Changes in membrane viscoelasticity due to bound C3d were not enhanced by bound C4b, C4d, C5, factor Bb or p; presence on RBC membrane of the latter two complement components may partially reverse the effect of C3d fixation. Lipid fluidity of RBC membrane, examined by fluorescence depolarization, increased with fixation of all complement components except C5. These complement-induced changes in membrane viscoelastic properties have potential pathophysiological and clinical implications. The data suggest that extravascular sequestration of human RBCs may be explained in part by increased membrane rigidity resulting from C3d fixation.     

Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent M(r) of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E(hu) membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E(s)) and released (125)I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E(hu) membrane protein was not altered by removal of sialic acid from the E(s) bearing C3b,Bb,P sites. E(hu) membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the alpha polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E(hu) membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.     

Chloride channels in normal and cystic fibrosis human erythrocyte membrane

Electrophysiological studies on human RBCs have been difficult due to fragility and small size of cells, and little is known of ionic conductive pathways present in the RBC membrane in health and disease. We report on anionic channels in cells of healthy donors (control) and cystic fibrosis (CF) patients. Anion channel activity (8-12 pS, linear) was induced in cell-attached configuration by forskolin (50 microM) and in excised inside-out configuration by PKA (100 nM) and ATP (1 mM) but control and CF RBCs differed by their respective kinetics and gating properties. These channels were permeable to ATP (100 mM, symmetrical Tris-ATP). These data suggest either the existence of two different anionic channel types or regulation of a single channel type either by the CFTR (cystic fibrosis transmembrane regulator) protein or by different cytosolic factors. Another anionic channel type displaying outward rectification (approximately 80 pS, outward conductance) was present in 30% of CF cell patches but was not observed in normal cell patches. The frequently recorded activity of this channel in CF patches suggests a down-regulation in normal RBCs.     

Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which the erythrocytes are abnormally sensitive to lysis by complement. A functional deficiency of membrane-associated complement regulators has been demonstrated on PNH erythrocytes. The two factor H-like proteins, the C3b receptor (CR1) and the decay-accelerating factor (DAF), were isolated from normal human erythrocytes, and specific antisera were prepared. Selective inhibition of the two proteins on normal erythrocytes by the antisera demonstrated (i) that the factor responsible for accelerated decay of erythrocyte-bound C3 convertase is DAF and (ii) that the cofactor required for inactivation of erythrocyte-bound C3b by factor I is CR1. PNH erythrocytes were deficient in both of these activities. Erythrocytes deficient in CR1, which were obtained from an apparently healthy individual, exhibited normal DAF activity but no factor I cofactor activity. These cells were not susceptible to complement-mediated lysis in acidified human serum, whereas PNH erythrocytes and Pronase-treated human erythrocytes (which lack DAF and CR1 activities) were lysed by this treatment. It is suggested that the protein primarily responsible for preventing complement activation on normal human erythrocytes is DAF. AMr 73,000 protein isolated from the normal erythrocyte membranes of one PNH patient by using anti-DAF IgG was largely absent from the abnormal erythrocytes of this individual, suggesting that PNH cells lack the DAF protein. CR1 antigen, however, was present on the abnormal PNH erythrocytes. The results suggest that the primary molecular defect underlying the clinical manifestations of PNH may be the lack of the membrane-associated DAF protein and that the abnormal cells may also exhibit impaired CR1 function.     

Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase

An ADP-ribosyltransferase was purified approximately 500-fold from the supernatant fraction of turkey erythrocytes. The enzyme hydrolyzed [carbonyl-(14)C]NAD to ADP-ribose and [carbonyl-(14)C]nicotinamide at a low rate. Nicotinamide formation from NAD was enhanced by arginine methyl ester > D-arginine approximately L-arginine > guanidine; lysine, histidine, and citrulline were ineffective. Incubation of [adenine-U-(14)C]NAD and arginine methyl ester or arginine with the purified enzyme resulted in the formation of new compounds that contained (14)C, reacted with ninhydrin, and quenched background fluorescence of thin-layer plates viewed in ultraviolet light. Their mobilities on thin-layer chromatograms were indistinguishable from those of ADP-ribosylarginine methyl ester and ADP-ribosylarginine formed during incubation of choleragen with NAD and arginine methyl ester or arginine, respectively [Moss, J. & Vaughan, M. (1977) J. Biol. Chem. 252, 2455-2457]. The purified transferase also catalyzed the incorporation of label from [adenine-(14)C]-NAD into lysozyme, histones and polyarginine. When the (14)C-labeled lysozyme was incubated with snake venom phosphodiesterase, the radioactivity was released and, on thin-layer chromatograms, exhibited a mobility indistinguishable from that of 5'-AMP, as would be expected of an ADP-ribosylated protein, but not of a poly(ADP-ribosylated) product. The purified transferase activated rat brain adenylate cyclase and, as is the case with choleragen, activation was absolutely dependent on NAD. The presence in the avian erythrocyte of a protein that, like choleragen and Escherichia coli heat-labile enterotoxin, apparently activates adenylate cyclase and possesses ADP-ribosyl transferase activity is consistent with the view that the mechanisms through which the bacterial toxins produce pathology are not entirely foreign to vertebrate cells, at least some of which may possess and employ an analogous mechanism for activation of adenylate cyclase.     

Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates.     

Isolation of the JMH antigen on a novel phosphatidylinositol-linked human membrane protein.

JMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospholipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI-linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.     

Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton.

Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of non-erythroid tissues. In mammalian erythrocytes, it plays a key role in regulating membrane physical properties of mechanical stability and deformability by stabilizing spectrin-actin interaction. We report here the molecular cloning and characterization of human erythrocyte protein 4.1 cDNA and the complete amino acid sequence of the protein derived from the nucleotide sequence. Probes prepared from the cloned erythrocyte protein 4.1 cDNA hybridized with distinct mRNA species from a wide variety of non-erythroid tissues, including brain, liver, placenta, pancreas, and intestine, implying substantial homology between erythroid and non-erythroid protein 4.1. The availability of cloned erythrocyte protein 4.1 cDNA should facilitate the study of the functional characteristics of this protein in erythroid as well as non-erythroid cells.     

Complete amino acid sequence and homologies of human erythrocyte membrane protein band 4.2.

The complete amino acid sequence for human erythrocyte band 4.2 has been derived from the nucleotide sequence of a full-length 2.35-kilobase (kb) cDNA. The 2.35-kb cDNA was isolated from a human reticulocyte cDNA library made in the expression vector lambda gt11. Of the 2348 base pairs (bp), 2073 bp encode 691 amino acids representing 76.9 kDa (the SDS/PAGE molecular mass is 72 kDa). RNA blot analysis of human reticulocyte total RNA gives a message size for band 4.2 of 2.4 kb. The amino acid sequence of band 4.2 has homology with two closely related Ca2(+)-dependent cross-linking proteins, guinea pig liver transglutaminase (protein-glutamine gamma-glutamyltransferase; protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) (32% identity in a 446-amino acid overlap) and the a subunit of human coagulation factor XIII (27% identity in a 639-amino acid overlap), a transglutaminase that forms intermolecular gamma-glutamyl-epsilon-lysine bonds between fibrin molecules. The region of greatest identity includes a 49-amino acid stretch of band 4.2, which is 69% and 51% identical with guinea pig liver transglutaminase and the a subunit of factor XIII, respectively, within the regions that contain the active sites of these enzymes. Significantly, within the five contiguous consensus residues of the transglutaminase active site, Gly-Gln-Cys-Trp-Val, band 4.2 has an alanine substituted for cysteine (which is apparently essential for activity). Consistent with this active site substitution, erythrocyte membranes or inside-out vesicles, which contain band 4.2, show no evidence of transglutaminase activity by two types of in vitro assay.     

Syndeins: the spectrin-binding protein(s) of the human erythrocyte membrane.

Two-dimensional tryptic and chymotryptic analyses of all the major bands in a sodium dodecyl sulfate/polyacrylamide gel of the human erythrocyte membrane show that each band has a characteristic map. However, band 2.1 (nomenclature of T. L. Steck) and several polypeptides below this band exhibit similar tryptic and chymotryptic peptide maps and thus appear to be a family of closely related proteins or degradation products. Furthermore, they all contain a subset of peptides that are accounted for by the peptides from two known spectrin-binding fragments. We show that both fragments derive from 2.1-related proteins and conclude that band 2.1 and its related proteins, which we name "syndeins", bind spectrin and connect it to the erythrocyte membrane.     

CD59 (homologous restriction factor 20), a plasma membrane protein that protects against complement C5b-9 attack, in human atherosclerotic lesions.

Blood cells express a cell membrane protein, termed homologous restriction factor 20 (HRF20) and identical to CD59, that can inhibit complement C5b-9 insertion into their membranes. In this report, we investigated by immunohistochemistry whether CD59 was present on cells in human atherosclerotic lesions since membranous C5b-9(m) has been found in lesions. Using a monoclonal anti-CD59 antibody, a cellular CD59 staining pattern was apparent in nearly all lesion specimens. CD59 stain co-localised with macrophage (CD14), T lymphocyte (CD7), endothelial cell (anti-factor VIII related antigen) and smooth muscle cell cytoskeletal-specific antigens (anti-alpha actin and muscle myosin). Endothelial cells always exhibited a more intense stain than the other cell types. CD59 antigen was not localised to any one area of the lesions. Usually CD59-positive cells occurred in clusters rather than as randomly spaced individual cells. CD59 did not stain all cells of the lesion and in particular did not appear to stain all smooth muscle cells. Areas of CD59-negative cells were sometimes observed to exhibit a cellular C5b-9 staining pattern. C5b-9 deposits were also observed in CD59-positive regions. Normal saphenous vein stained strongly for CD59 at the endothelial lining and weakly in the media. Capillaries in atherosclerotic intima always stained strongly for CD59. We conclude that HRF20 is constitutively expressed on endothelium and is under regulatory control in smooth muscle cells. Cellular C5b-9 attack in atherosclerotic lesions is therefore most likely to occur on smooth muscle cells.     

Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol-reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.     

Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm.

The venom of honeybees, Apis mellifera, contains several biologically active peptides and two enzymes, one of which is a hyaluronidase. By using degenerate oligonucleotides derived from the amino-terminal sequence of this hyaluronidase reported by others, clones encoding the precursor for this enzyme could be isolated from a cDNA library prepared from venom glands of worker bees. The deduced amino acid sequence showed that bee venom hyaluronidase is a polypeptide composed of 349 amino acids containing four cysteines and three potential sites for N-glycosylation. The sequence of the precursor also indicated that the conversion of the pro-enzyme to the end product must involve cleavage of a Thr-Pro bond, a most unusual processing reaction. The mRNA encoding hyaluronidase could also be detected in testes from drones. Expression of the cloned cDNA in Escherichia coli yielded a 41-kDa polypeptide that had hyaluronidase activity. Interestingly, the hyaluronidase from bee venom glands exhibited significant homology to PH-20, a membrane protein of guinea pig sperm involved in sperm-egg adhesion. These structural data support the long-held view that hyaluronidases play a role in fertilization.     

X-ray diffraction studies of human erythrocyte membrane structure.

Small angle x-ray diffraction patterns have been obtained from ordered arrays of hemoglobin-free human erthyrocyte membranes by use of improved techniques. Diffraction data have been recorded to 9 A resolution on samples whose lattice periodicity was varied (by changing humidity) from 55.5 A to 69.6 A. The observed reflections permitted tracing the intensity transform of the membranes. Phases for the reflections were assigned by the minimum wavelength principle. An electron density profile was then obtained by Fourier inversion, and yielded a symmetric membrane about 55 A in width. This structure can account for the previously reported diffuse scattering observed in other preparations (thus rendering unnecessary the proposed assignment of this scattering to a separated lipoprotein phase) and for the continuous scattering that we have recorded from isolated membranes in buffer. Lower resolution data that we have obtained from ultracentrifugally prepared lattices in buffer (and therefore without dehydration) are consistent with the above results, and support our view that we are observing diffraction from intact membranes.     

Impaired expression of erythrocyte glycosyl-phosphatidylinositol-anchored membrane CD59 in patients with psoriatic arthritis. Relation to terminal complement pathway activation

OBJECTIVE: Complement-mediated injury is regulated by many factors; among these CD59 has been identified as a widely distributed glycoprotein that inhibits membrane C5b-9 (terminal complement component) formation. The aim of the study was to assess erythrocyte CD59 expression in patients with psoriatic arthritis in order to understand the role of CD59 in the pathogenesis. METHODS: Washed erythrocytes from 50 patients with psoriatic arthritis, 8 with cutaneous psoriasis and 24 healthy subjects were incubated with monoclonal anti-CD59 antibody followed by a second FITC conjugated antibody and fluorescence intensity analysed by FAC-Scan flow cytometer to assess their CD59 membrane expression. SC5b-9 levels were measured in the plasma by ELISA and results compared with CD59 values. Immune complexes, complement C3 and C4 and rheumatoid factor were also determined. RESULTS: Impaired expression of erythrocyte membrane-anchored CD59 was found in patients with psoriatic arthritis; the lowest levels were seen in active patients (p < 0.01). Increased SC5b-9 was seen in the plasma of patients with active disease. An inverse correlation was also found between plasma C5b-9 and the CD59 expression levels (r = -0.81, p < 0.001). CONCLUSION: The low CD59 expression on erythrocytes from patients with psoriatic arthritis may be an index of a low tissue CD59 expression. This impairment could facilitate the activation of complement pathway and increase the risk for arthritis. Membrane attack complex formation in deficient membrane bound CD59 may also exacerbate synovial cell injury and inflammation.     

Plasmodium falciparum erythrocyte membrane protein 1 functions as a ligand for P-selectin.

The malarial protein Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a parasite protein that is exported to the surface of the infected erythrocyte, where it is inserted into the red cell cytoskeleton in the second half of the parasite life cycle. The surface expression of PfEMP1 coincides with the occurrence of the adhesion of infected erythrocytes to vascular endothelium. This protein has been shown to interact with CD36, intercellular adhesion molecule-1 (ICAM-1) and chondroitin sulfate A (CSA). In this study, it is demonstrated by affinity purification and western blot analysis that PfEMP1 also functions as a cell surface ligand for P-selectin, an adhesion molecule that has been shown to mediate the rolling of infected erythrocytes under physiologic flow conditions, leading to a significant increase in adhesion to CD36 on activated platelets and microvascular endothelium.     

Neoantigens of the membrane attack complex of human complement.

The membrane attack complex of complement is a fusion product of five complement proteins: C5b, C6, C7, C8, and C9. The complex causes complement-dependent cell membrane damage. It is assembled following complement activation both on the target cell surface and in the fluid phase. The isolated soluble complex, which has a molecular weight of one million, exhibited reduced expression of the antigenic determinants of the native precursor proteins. Antisera produced to the intact complex contained antibodies to neoantigens which were not detectable on the five precursor proteins. Antisera were rendered neoantigen-specific by adsorption with fresh human serum. Since the adsorbed antisera precipitated the complex, the complex must contain multiple neoantigenic sites. The complex-specific antibodies not only reacted with the soluble complex, but also with the target cell-bound membrane attack complex.     

Effect of transmembrane ion gradients on Raman spectra of sealed, hemoglobin-free erythrocyte membrane vesicles.

Sealed hemoglobin-free erythrocyte vesicles have been isolated. Imposition of transmembrane cation gradients increases the intensity of Raman scattering in the CH3-stretching region as observed with unsealed ghosts at temperatures greater than 38 degrees C and pH less than 7.0 [Verma, S. P. & Wallach, D. F. H. (1976) Proc. Natl. Acad. Sci. USA 73, 3358--3561]. Modifications in the amide I and amide III frequencies consistent with increased helicity of membrane proteins are observed upon imposition of a cation gradient. Spectrin-free vesicles also demonstrate cation gradient-sensitive intensity changes in the CH3-stretching region. However, no evidence for cation gradient-related protein conformation changes is found with these vesicles. The transmembrane potential of these vesicles has been altered by variations in anion composition and the electrogenic activity of Na+,K+-ATPase. The membrane potential was monitored by cyanine dye fluorescence. Imposition of a membrane potential (negative inside) also increased the intensity of Raman scattering in the CH3-stretching region. These results suggest that a transmembrane potential (negative inside) and/or cation gradient can energize membranes by compression of the apolar region and transfer of protein methyl residues into polar regions.