Human Red Cell Membrane Proteins Research Articles

The state of DNA methylation in the promoter regions of the human red cell membrane protein (band 3, protein 4.2, andβ-spectrin) genes

The state of methylation of the 5′-CpG-3′ sites is known to be linked to the regulation of promoter function by modulating DNA-protein interactions and to the structure of chromatin. As part of a project to determine methylation patterns in the human genome, the methylation profiles were examined in genes for the human erythroid membrane proteins; protein 4.2 (P4.2), gene (ELB42), band 3 (B3), gene (EPB3), and β-spectrin (β-Sp), gene (SPTB). The bisulfite protocol of the genomic sequencing method was applied.(1) In the DNA from peripheral white blood cells, the promoter regions of EPB3 and ELB42 were extensively methylated, but the promoter of SPTB was totally unmethylated. (2) During erythroid differentiation, (i) ELB42 was unmethylated in DNAs from the cell line UT-7/EPO, but became methylated (55−93 %) in cultured erythroblasts from peripheral BFU-E. The mRNA from ELB42 was first detected in early erythroblasts and protein 4.2 was expressed in late erythroblasts. (ii) EPB3 was consistently methylated in UT-7/EPO and also in cultured erythroblasts from burst forming unit erythroid (BFU-E) from peripheral blood. EPB3 and ELB42 were efficiently transcribed in UT-7 cells only after erythropoietin stimulation. (iii) SPTB remained unmethylated in DNAs from UT-7/EPO and cultured erythroblasts. (3) We also investigated methylation profiles in peripheral white blood cells from patients with erythroid diseases, like complete P4.2 deficiency due to ELB42 mutations, hereditary spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. The methylation profiles of the promoter regions of these three genes were essentially identical to those in healthy individuals.

Steric Immobilization of Liposomes in Chromatographic Gel Beads and Incorporation of Integral Membrane Proteins into Their Lipid Bilayers

Interactions between water-soluble substances and immobilized liposomes or proteoliposomes can be analyzed by chromatographic procedures. Methods were therefore developed to sterically immobilize large amounts of liposomes in gel beads. First, freeze-thawing of moist Sephacryl S-1000 beads mixed with egg yolk phospholipid liposomes of 200-300 mM lipid concentration immobilized liposomes representing 60-90 μmol lipid with an internal volume of 60-150 μl per milliliter gel bed. The same procedure with freeze-dried beads immobilized liposomes representing more lipid but with lower internal volumes. Other lipid mixtures could also be used. Second, mixtures of Sephacryl S-1000 beads and liposomes were freeze-dried and rehydrated. Large amounts of liposomes became immobilized even with dilute liposome suspensions. Third, a mixture of diethylether-dried Sephacryl S-1000 beads and an emulsion of an aqueous solution in diethylether with lipids was subjected to reverse-phase evaporation. Liposomes of high specific internal volume became immobilized. Integral human red cell membrane proteins could be incorporated into the liposomal lipid bilayers. Upon mixing octyl glucoside-solubilized proteins with moist beads containing immobilized liposomes, up to 0.4 mg protein became incorporated per milliliter gel bed (yield 34%). Proteins could also be incorporated during immobilization by reverse-phase evaporation. Detergents could be removed efficiently from immobilized (proteo)liposomes in a gel bed.

Conformational changes in human red cell membrane proteins induced by sugar binding

We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.

Topography of protein kinase C substrates analyzed by membrane splitting

We have used the methods of planar cell and membrane monolayer formation and monolayer splitting to study structural details of the transmembrane signaling process mediated by protein kinase C. We analyzed human red cell membrane proteins phosphorylated by phorbol ester activation of protein kinase C. Planar single membrane preparations, extraction procedures, and gel electrophoresis coupled with silver staining and autoradiography confirmed that two bands in the 100 kDa region, and bands 4.1, and 4.9, were peripheral and phosphorylated by treatment with 12- O-tetradecanoylphorbol 13-acetate (TPA). TPA also stimulated minor incorporation of [ 32P]P i into most integral membrane proteins, including band 3, glycophorin A, the band 4.5 region (glucose transporter) and band 7. Planar cell and membrane-splitting methods revealed that neither integral nor peripheral phosphorylated polypeptides were cleaved by freeze fracture, that all phosphorylated peripheral proteins partitioned intact with the cytoplasmic side of the membrane, and that the percentages of [ 32P]P i-labeled peripheral proteins were the same in split membrane cytoplasmic leaflets as in intact membranes. As a unique approach to examining protein topographies membrane splitting provides strong evidence that the major phosphorylated products of the polyphosphatidylinositide pathway are topographically associated with the cytoplasmic leaflet of the human erythrocyte plasma membrane. We further conclude that TPA-induced phosphorylation of red cell peripheral proteins does not significantly alter their transbilayer partitioning patterns after membrane splitting.

Fractionation of human red cell membrane proteins by ion-exchange chromatography in detergent on mono Q, with special reference to the glucose transporter

The anion exchanger Mono Q has been used for rapid and efficient fractionation of human red cell membrane proteins in the easily removable detergents n-octyl-β- d-glucopyranoside or nonanoyl-N-methylglucamide. In practice the chromatographic resolution of membrane proteins was lower than for water-soluble proteins, perhaps due to protein—protein interactions and microheterogeneity, but several components, or groups of components, separated well upon salt gradient elution. The glucose transporter (or transportase) was eluted early, glycophorins later, and the anion transporter still later. The detergents Berol 185 and the zwitter-ionic derivatives of cholate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulphonate, gave similar chromatographic results but differed in solubilization selectivity. A relatively pure material was also fractionated; viz., a glucose transportase which had been prepared by DEAE-cellulose chromatography. Mono Q, in the presence of octyl glucoside, afforded additional purification, which made automatic sequence determination possible for eighteen amino acid residues. The results indicate that two polypeptides were present in about equimolar amounts.

Optimized procedures for investigating changes in human erythrocyte membrane proteins using treatment with calcium and various proteases

In order to establish reproducible sensitive techniques for investigating diseases in which membrane defects have been implicated, human red cell membrane proteins were studied by SDS electrophoresis in ghosts prepared from the blood of 35 separate adult donors using treatment with 1 mmol/1 calcium and proteolytic dissection under standardized conditions. The effects of calcium were consistent with increased binding of cytosolic proteins in membranes from fresh blood and endogenous proteolysis in membranes from aged blood. Exogenous proteolysis was most effective with trypsin and was facilitated both by pre-binding this protease to the membrane and by inclusion of 1 mmol/1 Ca 2+. The nature and extent of these effects was highly dependent on the storage time of the blood. Preliminary indications of cleavage fragment length polymorphism was obtained after treatment with the S. aureus V8 protease but not after trypsin or a chymotrypsin treatment.

Membrane proteins synthesized by human reticulocytes and their precursors.

Membrane protein synthesis in human immature erythroid cells was studied by incubating the cells with 35S-methionine in vitro. The radioactive precursor amino acid was incorporated into membrane protein in a linear fashion for approximately 60 min, after which there was only a slight increase in incorporation. Intracellular protein synthesis, in contrast, was linear for periods up to 2 h. Analysis of isolated membranes by polyacrylamide gel electrophoresis in sodium dodecyl sulphate showed that peripheral blood reticulocytes synthesized two proteins in the 4.5 region (MW=50-60000 D) and a third protein coinciding with band 6 (glyceraldehyde-3-phosphate dehydrogenase). Separation of reticulocytes into different age groups on stractan II gradients showed more immature reticulocytes synthesize a wider range of membrane proteins, extending from bands 4.1 to 8. When nucleated red cells were present in the incubations, synthesis of band 3 was also observed. Earlier erythroid precursor present in erythropoietic BFU-E cultures synthesized spectrin in addition to the other membrane proteins. The data indicate that human red cell membrane protein synthesis follows a programmed pattern and that as the erythroid elements mature they lose the capacity to synthesize certain membrane proteins.

Rapid quantitative separation of the major glycoproteins (PAS 1, 2, and 3) from other human red cell membrane proteins in a nondenaturing medium by affinity chromatography

Several methods were tested for separating the major glycoproteins from other proteins extracted from human red cell membranes by the nondenaturing detergent, Triton X-100. Separation could be achieved by isoelectric focusing, but the intrinsic proteins (predominantly band 3) become irreversibly precipitated. Dithiol-containing resin (Thiol-Sepharose, Pharmacia) was found to be capable of removing the glycoproteins [Kahlenberg, A. (1976) Anal. Biochem. 74, 337–342], but the procedure is relatively slow. Rapid and quantitative separation was achieved with an organomercurial gel (Affi-Gel 501, Bio-Rad). The major glycoproteins were not retarded, but all of the other membrane proteins were retained in the gel. They could be subsequently eluted with solutions containing cysteine.

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS).

DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.

Effects of pH during recombination of human erythrocyte membrane apoprotein and lipid

The recombinates from human red cell membrane proteins and lipids resulting from dialysis of the components in 2-chloroethanol against aqueous buffers from pH 2–12 have been studied by density gradient centrifugation, polyacrylamide gel electrophoresis and freeze-fracture electron microscopy. Between pH 4 and 10 most of the proteins were found in the recombinates whereas below pH 4 and above pH 10 only part of them were recovered in the lipoprotein band after density gradient centrifugation. At low pH, increasing incorporation of the “major glycoprotein” into the recombinates was detected by gel electrophoresis and in parallel increasing amounts of particles were found in the freeze-fractured membrane faces. The necessity of working at low pH values from pH 2–4, however, and a critical evaluation of all the data presently available leads to the conclusion that the 2-chloroethanol technique is not adequate for recombination studies tending to membrane reconstitution.

Extraction of erythrocyte membrane proteins by sulfhydryl inhibitors.

Human red cell membrane proteins were extracted by incubation of the ghost with hypotonic phosphate buffer (pH 7.4), N-ethylmaleimide and p-hydroxy-mercuribenzoate. In paroxysmal nocturnal hemoglobinuria (PNH), hereditary spherocytosis (HS) and hereditary elliptocytosis, the amount of proteins extracted by these procedures was significantly less than the amount extractable from the ghost of normal and aplastic anemia red cells. Polypeptide patterns of red cell membranes in these hematological disorders were essentially similar to those of normal ghosts. Analysis of the supernatant by SDS polyacrylamide gel electrophoresis revealed that this reduction was mainly due to the reduced amount of peripheral proteins extracted. The extraction of peripheral proteins by sulfhydryl reagents was accompanied by shape changes resulting in the formation of membrane vesicles, suggesting an important role of peripheral proteins in the maintenance of ghost shape. It is also suggested that qualitative abnormalities of peripheral proteins such as altered reactivity to sulfhydryl reagents and/or strong binding to the membrane are present in PNH, HS and hereditary elliptocytosis red cells.

Immunochemical studies of human red cell membrane proteins excreted in normal urine

Immunochemical studies of human red cell membrane proteins excreted in normal urine

Immunochemical studies of human red cell membrane proteins excreted in normal urine

Two red cell membrane antigens are detected by immunochemical techniques in normal human urine. Antibodies against can be obtained by immunizing rabbits with total red cell membrane protein fraction, purified red cell membrane glycoproteins or with immune precipitate of the latter antiserum and normal urine. With these reagents the two urinary antigens can be demonstrated on the surface of red cells of any major blood group specificity. One urinary component is antigenically related to the main membrane glycoprotein, whereas the other is linked to a different membrane protein and, furthermore, is not released when red cells are treated with trypsin. A study of the nature and the distribution of this antigen on the red cell membrane is presented.

Recombination of human red cell membrane protein fractions with homologous lipids

Human red cell membrane proteins were separated from membrane lipids by butanol-water partition, solubilized in sodium dodecyl sulfate and fractionated by gel filtration on a Sepharose 4B column. the eluates were pooled in two fractions: Fraction I containing the high molecular weight proteins, around 200 000, and Fraction II comprising the proteins which migrate as 100 000 and less. Fractions I and II had different sialic acid content and pH-dependent solubility. Both protein fractions recombined with red cell lipids when mixtures in sodium dodecyl sulfate were dialyzed against aqueous buffers. The amount and composition of recombinate obtained at various pH values were different for the two fractions. The recombinates obtained at pH 3.6 from Fraction I and from Fraction II had different density and stability towards change in pH and ionic strength. The recombinates of both fractions exhibited a membrane-like structure on electron microscopy.