Modification of glycophorin A during oxidation of erythrocyte membrane

Human erythrocyte ghosts were oxidized with tert-butyl hydroperoxide and subsequently treated with tritiated borohydride to label the membrane proteins modified during the membrane oxidation. From the ghosts, oxidized-and-tritiated glycophorin A was isolated and characterized. No intermolecular cross-links were observed as analyzed by sodium dodecylsulfate gel electrophoresis. But, the number of lysine residues was significantly reduced and susceptibility to proteinases such as trypsin, chymotrypsin and pronase was lower than that of control glycophorin A. Trypsinization of the oxidized-and-tritiated glycophorin A gave insoluble and soluble trypsin fragments. After dansylation, N-terminal amino acids of the trypsin-fragments were determined. Dansyl amino acids from the insoluble trypsin fragments were not identical with those from control insoluble counterparts in the membrane-spanning region of glycophorin A molecule. Fractionation by gel filtration of dansyl-soluble trypsin fragments, and the N-terminal amino acid analysis of the fractionated peptides indicated that the peptides derived from the glycosylated region located in the outside of the membrane matrix were identical with those from control soluble counterparts. The results suggest that the glycosylated outside region of glycophorin A was modified only slightly but the hydrophobic membrane-spanning region was extensively modified during membrane oxidation, most likely by oxidized lipids.     

A single mutation in the glycophorin A binding site of hepatitis A virus enhances virus clearance from the blood and results in a lower fitness variant

Hepatitis A virus (HAV) has previously been reported to bind to human red blood cells through interaction with glycophorin A. Residue K221 of VP1 and the surrounding VP3 residues are involved in such an interaction. This capsid region is specifically recognized by the monoclonal antibody H7C27. A monoclonal antibody-resistant mutant with the mutation G1217D has been isolated. In the present study, the G1217D mutant was characterized physically and biologically in comparison with the parental HM175 43c strain. The G1217D mutant is more sensitive to acid pH and binds more efficiently to human and rat erythrocytes than the parental 43c strain. In a rat model, it is eliminated from serum more rapidly and consequently reaches the liver with a certain delay compared to the parental 43c strain. In competition experiments performed in vivo in the rat model, the G1217D mutant was efficiently outcompeted by the parental 43c strain. Only in the presence of antibodies reacting specifically with the parental 43c strain could the G1217D mutant outcompete the parental 43c strain in serum, although the latter still showed a remarkable ability to reach the liver. Altogether, these results indicate that the G1217D mutation induces a low fitness phenotype which could explain the lack of natural antigenic variants of the glycophorin A binding site.     

Synthesis of a docosapeptide comprising the hydrophobic membrane spanning region of glycophorin A

The docosapeptide which constitutes the membrane spanning region (amino acid residues 73-94) of the human red blood cell protein glycophorin A has been synthesized. This may be the first example of the synthesis of the entire membrane embedded domain of a membrane spanning protein. Three fully protected fragments were prepared by stepwise elongation using dicyclohexylcarbodiimide and p-nitrophenyl ester activation of N alpha-tert.-butyloxycarbonyl amino acids. The three fragments represent amino acid residues 73-79, 80-86, and 87-94 in the sequence of glycophorin A and contain a large proportion of valine, leucine, and isoleucine residues but contain no amino acids with ionizable side chain functional groups. The three fragments were condensed using both the azide method and the dicyclohexylcarbodiimide method to give fully protected docosapeptide. Benzyl groups protecting the side chains of the docosapeptide were removed by prolonged hydrogenolysis to give the desired product N alpha-tert.-butyloxycarbonyldocosapeptide ethyl ester. High resolution proton n.m.r. spectra of the protected fragments in 100% deuterochloroform showed all resonances to be broadened with the amide resonances broadened beyond recognition. In perdeuterodimethylsulfoxide all resonances were relatively sharp with all amide resonances visible and showing coupling constants of 7-8 Hz. Solvent titration of the proton spectra of two of the fragments from 100% perdeuterodimethylsulfoxide to 100% deuterochloroform demonstrated a transition to the broadened spectrum, accompanied by a decrease in the coupling constant of the amide protons (JNH-CH alpha) suggesting solvent dependent onset of intramolecular secondary structure, possibly accompanied by aggregation. A proton n.m.r. spectrum of the docosapeptide in perdeuterodimethylsulfoxide shows a few resolved amide resonances with coupling constants of 7-9 Hz. Solvent titration with perdeuterochloroform again suggests a transition to a rigid intramolecular secondary structure.     

Features of organic nonelectrolyte binding to the erythrocyte membrane

The capacity of erythrocyte membranes for organic nonelectrolytes from different chemical groups of chemical compounds was studied by the spin probe method and scanning electron microscopy. Hydrophobic spin-labeled derivatives of gamma-carbolin and stearic acid and the screened phenol antioxidant fenozan-1 were used as nonelectrolytes. Based on the analysis of electron spin resonance spectra of the hydrophobic spin-labeled nonelectrolytes and electron micrographs of erythrocytes, differences in the capacity of distribution regions in the intramembrane space of the derivative of gamma-carbolin and fenozan-1, on the one hand, and the spin-labeled derivative of fatty acid, on the other hand, were found. The first group has at least two membrane distribution regions, whereas in the second case only one type of distribution was found. The influence of limited membrane capacity on the realization of biological activity of organic nonelectrolytes is discussed.     

Nitrobenzylthioinosine binding sites in the erythrocyte membrane

Nitrobenzylthioinosine binds tightly, but reversibly, to sites in the human erythrocyte membrane; occupancy of these sites blocks the transport of uridine and of other nucleosides. This report describes the inhibition of nitrobenzylthioinosine binding at these sites by substrates of the uridine transport mechanism and by compounds related to nitrobenzylthioinosine. For some of these compounds dissociation constant for binding at the nitrobenzylthioinosine sites were determined, assuming competition with nitrobenzylthioinosine.Deoxycytidine, a substrate for the uridine transport mechanism, did not inhibit binding of nitrobenzylthioinosine, suggesting that binding sites for the latter are distinct from nucleoside sites directly involved in transport.     

The hepatitis E virus polyproline region is involved in viral adaptation

Genomes of hepatitis E virus (HEV), rubivirus and cutthroat virus (CTV) contain a region of high proline density and low amino acid (aa) complexity, named the polyproline region (PPR). In HEV genotypes 1, 3 and 4, it is the only region within the non-structural open reading frame (ORF1) with positive selection (4-10 codons with dN/dS>1). This region has the highest density of sites with homoplasy values >0.5. Genotypes 3 and 4 show ～3-fold increase in homoplastic density (HD) in the PPR compared to any other region in ORF1, genotype 1 does not exhibit significant HD (p<0.0001). PPR sequence divergence was found to be 2-fold greater for HEV genotypes 3 and 4 than for genotype 1. The data suggest the PPR plays an important role in host-range adaptation. Although the PPR appears to be hypervariable and homoplastic, it retains as much phylogenetic signal as any other similar sized region in the ORF1, indicating that convergent evolution operates within the major HEV phylogenetic lineages. Analyses of sequence-based secondary structure and the tertiary structure identify PPR as an intrinsically disordered region (IDR), implicating its role in regulation of replication. The identified propensity for the disorder-to-order state transitions indicates the PPR is involved in protein-protein interactions. Furthermore, the PPR of all four HEV genotypes contains seven putative linear binding motifs for ligands involved in the regulation of a wide number of cellular signaling processes. Structure-based analysis of possible molecular functions of these motifs showed the PPR is prone to bind a wide variety of ligands. Collectively, these data suggest a role for the PPR in HEV adaptation. Particularly as an IDR, the PPR likely contributes to fine tuning of viral replication through protein-protein interactions and should be considered as a target for development of novel anti-viral drugs.     

Calcium binding by the erythrocyte membrane

Calcium binding to isolated erythrocyte membranes was stimulated by ATP. This stimulatory effect of ATP required Mg²⁺.Ethacrynic acid and ruthenium red inhibited the stimulatory effect of ATP.About 80% of the bound Ca²⁺ was associated with the membrane protein.The strongly bound Ca²⁺ was confined to two high molecular weight membrane proteins.Increasing amounts of Ca²⁺ bound to the membrane inhibited Na⁺ binding in the presence of ATP.     

Subunit structure of human erythrocyte glycophorin A.

Glycophorin A is a sialoglycoprotein isolated from human erythrocyte membranes which seems to exist as stable dimeric complexes in the presence of sodium dodecyl sulfate. When analyzed by dodecyl sulfate acrylamide electrophoresis this molecule forms two PAS-stainable bands (PAS-U and PAS-2) which are reversibly interconvertible. This change in electrophoretic mobility is dependent on the concentration of dodecyl sulfate, the use of Trisbuffer systems, the protein concentration in the incubation mixture, and the duration and temperature of incubation before electrophoresis. Reducing agents do no influence the results. Chromatography of the sialoglycopeptides on Sepharose columns in dodecyl sulfate before and after heat treatment gave similar results. A small hydrophobic peptide (T-6) derived from glycophorin A was able to prevent reassociation of the monomeric subunits back to the higher molecular weight form. This peptide was able to bind to the subunit of glycophorin A, but not to the high molecular weight complex. These results are consistent with a model of glycophorin A composed of two subunits which can dissociate and reassociate in the presence of detergents. These subunits may interact via the hydrophobic portions of the polypeptide chains.     

Nitrobenzylthionionosine binding sites in the erythrocyte membrane.

Nitrobenzylthioinosine binds tightly, but reversibly, to sites in the human erythrocyte membrane; occupancy of these sites blocks the transport of uridine and of other nucleosides. This report described the inhibition of nitrobenzylthioinosine binding at these sites by substrates of the uridine transport mechanism and by compounds related to nitrobenzylthioinosine. For some of these compounds dissociation constants for binding at the nitrobenzylthioinosine sites were determined, assumming competition with nitrobenzylthioinosine. Deoxycytidine, a substrate for the uridine transport mechanism, did not inhibit binding of nitrobenzylthioinosine, suggesting that binding sites for the latter are distinct from nucleoside sites directly involved in transport.     

Capsid assembly and involved function analysis of twelve core protein mutants of duck hepatitis B virus.

The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined. Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli. The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E. coli. The expressed core mutants without this region apparently inhibited E. coli growth. The results of transmission electron microscopy of E. coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E. coli. However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E. coli. In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein.     

Regulation of the glycophorin C-protein 4.1 membrane-to-skeleton bridge and evaluation of its contribution to erythrocyte membrane stability

The band 3-ankyrin-spectrin bridge and the glycophorin C-protein 4.1-spectrin/actin bridge constitute the two major tethers between the erythrocyte membrane and its spectrin skeleton. Although a structural requirement for the band 3-ankyrin bridge is well established, the contribution of the glycophorin C-protein 4.1 bridge to red cell function remains to be defined. In order to explore this latter bridge further, we have identified and/or characterized five stimuli that sever the linkage in intact erythrocytes and have examined the impact of this rupture on membrane mechanical properties. We report here that elevation of cytosolic 2,3-bisphosphoglycerate, an increase in intracellular Ca(2+), removal of cell O(2), a decrease in intracellular pH, and activation of erythrocyte protein kinase C all promote dissociation of protein 4.1 from glycophorin C, leading to reduced retention of glycophorin C in detergent-extracted spectrin/actin skeletons. Significantly, where mechanical studies could be performed, we also observe that rupture of the membrane-to-skeleton bridge has little or no impact on the mechanical properties of the cell, as assayed by ektacytometry and nickel mesh filtration. We, therefore, suggest that, although regulation of the glycophorin C-protein 4.1-spectrin/actin bridge likely occurs physiologically, the role of the tether and the associated regulatory changes remain to be established.     

A convenient erythrocyte membrane cyclic AMP binding assay.

Formaldehyde reacts with tryptophan in the presence of sulfuric acid and iron, nickel, or cobalt and gives a violet color with absorption maximum at 575 nm. Nanomoles of formaldehyde can be estimated spectrophotometrically by using this reaction which is also very specific. Other aminoacids, sugars and related compounds, higher aldehydes, and heavy metals do not react. 3-Substituted indoles give similar colors of less intensity. The tryptophan reaction can be used to measure formaldehyde formed from biochemical reactions using fractions of liver and other tissues.     

Exposure to ionizing radiation decreases hemoglobin binding to erythrocyte membrane

The influence of gamma-radiation at doses 10, 10(2) and 10(3) Gy on binding of hemoglobin to the erythrocyte membrane by inductive--resonance energy transfer in the donor-acceptor pair anthracene-hemoglobin was studied. It was found that binding of hemoglobin to the erythrocyte membrane decreases with increasing dose of gamma-radiation.     

Partial reversible inactivation of enzymes due to binding to the human erythrocyte membrane

Summary Hypotonic human erythrocyte ghosts, devoid of the original glyceraldehyde-3-phosphate dehydrogenase content of the red cell, bind added glyceraldehyde-3-phosphate dehydrogenases, isolated from human erythrocytes, rabbit and pig muscle, as well as rabbit muscle aldolase. There are only slight differences in the affinities towards the various glyceraldehyde-3-phosphate dehydrogenases. On the other hand, glyceraldehyde-3-phosphate dehydrogenases are bound much stronger than aldolase; in an equimolar mixture the former can prevent the binding of the latter, or replace previously bound aldolase at the membrane surface. Binding is always accompanied by the partial inactivation of enzymes, which can be reverted by desorption. Unwashed ghosts rich in hemoglobin seem to have a more pronounced inactivating effect on bound glyceraldehyde-3-phosphate dehydrogenase. In isotonic media ghosts, whether white or unwashed, reseal and do not interact with the enzymes.     

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the majorO-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey,Macaca fuscata) erythrocyte membranes were characterized.N-Glycolylneuraminic acid (neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. ThreeO-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose andN-acetylgalactosaminitol in the molar ratios of 111 (trisaccharide), 211 (tetrasaccharide) and 111 (pentasaccharide). The content of oligosaccharide units was estimated to be 1125 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc2-3Gal1-3[Neu5Gc2-6]GalNAcol.     

A cleavable N-terminal membrane anchor is involved in membrane binding of the Escherichia coli SRP receptor

Different from eukaryotes, the bacterial signal recognition particle (SRP) receptor lacks a membrane-tethering SRP receptor (SR) β subunit and is composed of only the SRα homologue FtsY. FtsY is a modular protein composed of three domains. The N- and G-domains of FtsY are highly similar to the corresponding domains of Ffh/SRP54 and SRα and constitute the essential core of FtsY. In contrast, the weakly conserved N-terminal A-domain does not seem to be essential, and its exact function is unknown. Our data show that a 14-amino-acid-long positively charged region at the N-terminus of the A-domain is involved in stabilizing the FtsY-SecYEG interaction. Mutant analyses reveal that the positively charged residues are crucial for this function, and we propose that the 14-amino-acid region serves as a transient lipid anchor. In its absence, the activity of FtsY to support cotranslational integration is reduced to about 50%. Strikingly, in vivo, a truncated isoform of FtsY that lacks exactly these first 14 amino acids exists. Different from full-length FtsY, which primarily cofractionates with the membrane, the N-terminally truncated isoform is primarily present in the soluble fraction. Mutating the conserved glycine residue at position 14 prevents the formation of the truncated isoform and impairs the activity of FtsY in cotranslational targeting. These data suggest that membrane binding and function of FtsY are in part regulated by proteolytic cleavage of the conserved 14-amino-acid motif.