Identification and partial characterization of the human erythrocyte membrane component(s) that express the antigens of the LW blood-group system.

Rhnull human erythrocytes lack the antigens of the Rhesus blood-group system, have an abnormal shape, have an increased osmotic fragility, and are associated with mild chronic haemolytic anaemia. Rhnull erythrocytes also lack all antigens of the LW blood-group system, but the functional significance of this deficiency is unknown. We have identified, by immunoblotting with two mouse monoclonal antibodies (BS46 and BS56), the LW-active component(s) in normal human erythrocytes as a broad band of Mr 37 000-47 000 on SDS/polyacrylamide-gel electrophoresis. Treatment of intact human erythrocytes with endoglycosidase F preparation destroyed the epitopes recognized by antibodies BS46 and BS56, suggesting that one or more N-glycosidically linked oligosaccharides are required for the formation of the LW antigens. Estimation of the number of LW antigen sites per erythrocyte by using radioiodinated purified antibody BS46 gave average values of 4400 molecules/cell for Rh(D)-positive adult erythrocytes and 2835 molecules/cell for Rh(D)-negative adult erythrocytes. Like the Rh(D) polypeptide, the LW polypeptide(s) is (are) associated with the cytoskeleton of normal erythrocytes. These results suggest the possibility that the absence of the LW polypeptide may also contribute to the functional and/or morphological abnormalities of Rhnull erythrocytes.     

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.     

Localization of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) antigens of human Rh blood groups in fetal erythrocyte membranes

The fetal erythrocyte membranes were partially solubilized with Triton X-100 at the low concentration (0.5%). The localizations of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) were investigated. Using hemagglutination inhibition assay, Rh1(D) antigen activity was observed in the Triton-treated membrane (Triton shell) containing mainly band 1, 2 (spectrin), band 5 (actin), band 4.1 and a part of band 3, while Rh2(C), 3(E), 4(c), 5(e) and 25(LW) antigens were detected in the supernatant containing band 3, 6, 2.2, 2.3 and 4.2. It is suggested that: Rh1(D) antigen would associate with cytoskeleton matrix of fetal erythrocyte membranes; Rh1(D) and Rh25(LW) antigens might be integral membrane proteins, while Rh2(C), 3(E), 4(c) and 5(e) antigens would be surface membrane proteins which are easily released from membranes by EDTA, mercaptoethanol and alkaline treatments.     

Absence of two membrane proteins containing extracellular thiol groups in Rhnull human erythrocytes.

Rhnull human erythrocytes lack all the antigens of the Rhesus blood-group system and are associated with mild chronic haemolytic anaemia. These erythrocytes have an abnormal shape and increased osmotic fragility. Labelling studies with the impermeant maleimide N-maleoylmethionine [35S]sulphone show that Rhnull erythrocytes lack two extracellular thiol-group-containing membrane components of apparent mol.wts. 32 000 and 34 000. Immunoprecipitation with mouse monoclonal antibody R6A (which reacts with all normal erythrocytes, but fails to react with Rhnull erythrocytes) specifically precipitates the 34 000-mol.wt. component from normal erythrocytes. Similar studies with human anti-Rh(D) serum shows that this antibody reacts with the 32 000-mol.wt. component. The results suggest that the R6A-binding polypeptide and the Rh(D) polypeptide may be involved in the maintenance of the shape and viability of the human erythrocyte.     

Demonstration of the activity of a rhesus-positive antigen in the erythrocytes of rhesus-negative donors and an increase in the expression of ABO system antigens after ultraviolet irradiation of the blood

UV irradiation (254 nm) in doses increasing erythrocyte (Er) hemolysis by 5 to 32% was found to stimulate the agglutination activity of ABO and Rhesus (Rh) system antigens. The stimulation effect was the higher the lower the antigen activity before irradiation. In the Rh-negative (Rh-)-Er, irradiation induced the Rh0(D)-like antigen specific activity suggesting that this antigen may be present in the Rh--Er membrane. Expression of Rh0(D)-antigen in Rh--Er, stimulation of its activity in Rh-positive cells, and activation of ABO system antigens may result from photo-chemical destruction of the outer membranous layer of the ER.     

The Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with [3H]palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.     

Functional and structural changes in the surface of human erythrocytes after irradiation with UV rays of various wave lengths. I. The expression of ABO and rhesus system antigens

UV irradiation (254 nm) in doses increasing erythrocyte haemolysis by 5, 10, 18 and 28 per cent was found to stimulate, by 2--16 times, the agglutination activity of ABO and Rh system antigens. The stimulation effect was the higher the lower the antigen activity before irradiation. In the Rh-negative (Rh-) erythrocytes, irradiation induced manifestation of the Rh0(D)-antigen specific activity suggesting that this antigen may be present in the Rh- erythrocyte membrane. The expression of Rh0(D)-antigen in Rh- erythrocytes, the stimulation of its activity in Rh-positive cells, and the activation of ABO system antigens may result from a photochemical destruction of the outer perimembraneous layer and release some of its components which stain in situ with alcian blue to be presumably glycoproteins. This effect is necessary to keep in mind when UV-irradiated blood transfusion is performed in therapeutic aims Rh- patients.     

Radioactive labeling techniques for the identification of G antigen in the human Rh blood group system

The G antigen is one of the erythrocyte membrane Rh antigens. The amount of Rh antigen present on the red blood cell is about 10(-15) g and radioactive labeling of membrane proteins is a useful method for its identification and characterization. In this paper, we compare 4 labeling techniques. Using a human monoclonal anti-Rh(G) antibody and an immunofixation technique, we located the G antigen on a polypeptide of an average molecular weight of 28,000 Da.     

Protein-sequence studies on Rh-related polypeptides suggest the presence of at least two groups of proteins which associate in the human red-cell membrane.

The Rh D blood-group antigen forms part of a complex, involving several other polypeptides, that is deficient in the red cells of individuals who lack all the antigens of the Rh blood-group system (Rhnull red cells). These include components recognized by anti-(Rh D) antibodies and the murine monoclonal antibodies R6A and BRIC 125. We have carried out protein-sequence studies on the components immunoprecipitated by these antibodies. Anti-(Rh D) antibodies immunoprecipitate an Mr-30,000-32,000 polypeptide (the D30 polypeptide) and an Mr-45,000-100,000 glycoprotein (D50 polypeptide). Antibody R6A immunoprecipitates two glycoproteins of Mr 31,000-34,000 (R6A32 polypeptide) and Mr 35,000-52,000 (R6A45 polypeptide). The D30 and R6A32 polypeptides were found to have the same N-terminal amino acid sequences, showing that they are closely related proteins. The D50 polypeptide and the R6A45 polypeptide also had indistinguishable N-terminal amino acid sequences that differed from that of the D30 and R6A32 polypeptides. The putative N-terminal membrane-spanning segments of the two groups of proteins showed homology in their amino acid sequence, which may account for the association of each of the pairs of proteins during co-precipitation by the antibodies. Supplementary data related to the protein sequence have been deposited as Supplementary Publication SUP 50417 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

A 220 kDa polypeptide, immunolocalized to epithelial tight junctions, is associated with brain clathrin preparations

Antibodies were raised in rabbits to highly purified preparations of bovine brain clathrin. The serum stained by immunofluorescence rat liver sections at tight junctions in a pattern that was identical to that previously reported (B. R. Stevenson et al.: J. Cell Biol. 103, 755-766 (1986] in which a monoclonal antibody specific to a 220 kDa (ZO-1) liver tight junction component was used. The serum also stained regions of the cell surface corresponding to the positions of intercellular junctions in confluent MDCK and HepG-2 cell cultures. Analysis of brain clathrin preparations resolved by polyacrylamide gel electrophoresis by immunoblotting with the serum indicated reaction with clathrin heavy and light chains as well as towards a 220 kDa polypeptide that was a minor component. Affinity purification of the serum provided antibodies directed mainly to clathrin light chains and these antibodies, as well as an independent antiserum to clathrin heavy chains, immunofluorescently stained liver tissue and cells in a manner typical of coated membranes/vesicles. These results suggested, by difference, that antibodies to a 220 kDa polypeptide, a minor constituent in brain clathrin preparations, were responsible for staining intercellular tight junctions in epithelia. The 220 kDa polypeptide present in brain clathrin preparations was demonstrated to be immunologically distinct from liver myosin heavy chain as well as erythrocyte and brain ankyrin. Comparison by two-dimensional mapping of the 220 kDa in brain clathrin with the clathrin heavy chain (180 kDa) polypeptide showed they were different proteins, but the 220 kDa polypeptide present in rat liver tight junctions was highly similar to the 220 kDa present in bovine brain clathrin preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen

Erythrocytes bearing the Rh(D) antigen have an Mr 30,000 integral membrane protein which can be surface-labeled with 125I and can be quantitatively immunoprecipitated from Triton X-100-solubilized spectrin-depleted membrane vesicles. The 125I-labeled Rh(D)-associated protein was purified to radiochemical homogeneity from membrane skeletons solubilized in sodium dodecyl sulfate and urea by hydroxylapatite chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The Rh(D)-associated protein was purified nearly 200-fold from 2 units of erythrocytes from DD individuals by employing similar methods on a large scale using the purified 125I-labeled Rh(D)-associated protein as a tracer. The product appeared to be greater than 95% pure and migrated as a diffuse band of Mr approximately 30,000-32,000 on silver-stained sodium dodecyl sulfate electrophoresis gels poured from 12% acrylamide. It is estimated that the Rh(D)-associated protein makes up approximately 0.5% of the original membrane protein. When concentrated, partially purified Rh(D)-associated protein forms dimers and larger oligomers which are stable in sodium dodecyl sulfate and urea. The Rh(D)-associated protein was protected from degradation when intact erythrocytes or inside out membrane vesicles were enzymatically digested. These studies indicate that the Mr 30,000 protein associated with the Rh(D) antigen is linked to the membrane skeleton, resides within the lipid bilayer with minimal extra- or intracellular protrusions, exists normally as an oligomer, and can be purified in denatured form.     

Quantitative comparison of Rh1 (Rho, D) antigen on D, Du and d red cells by radioimmunoassay using 125I-protein A

Relative Rh1 (Rho, D) antigen contents of the red Rh: 1 (Rh positive, D), Rh: wl (Rh variant, Du) and Rh: -1 (Rh negative, d) cells were estimated from the quantity of 125I-protein A bound to the sensitized red cells. The isotope binding activity to both D and Du cells decreased in parallel with the dilution of anti-D serum. The relative amount of the 125I-protein A bound to Du cells was about one-sixth that of D cells without papain treatment, while no isotope binding was observed in d cells. The Du red cells were quantitatively deficient in Rh1 (Rho, D) antigen activity compared with the D cells. A radioimmunoassay using 125I-protein A was a very useful method for studies regarding measuring the relative amounts of various blood group antigens.     

Erythrocyte autoantibodies induced in mice immunized with rat antigens.

Antibody reacting against syngeneic mouse erythrocytes could not be elicited (by rat erythrocytes) in athymic nude mice. Rat antigen preparations from heart, muscle testes, brain erythrocyte ghosts and foetal material failed to elicit detectable autoantibody reactivity against mouse erythrocytes, even though all these preparations (other than brain and foetal) induced a reduction in half life of syngeneic murine erythrocytes in vivo. We suggest that an unstable rat erythrocyte antigen is responsible for the induction of the autoimmune reactions, which in turn depend on T-cells for their generation.     

The identification of specific Rhesus-polypeptide-blood-group-ABH-active-glycoprotein complexes in the human red-cell membrane.

1. RhD,c and E immune complexes isolated from 3H- and 125I-surface-radiolabelled and unlabelled intact human red cells were analysed by SDS/polyacrylamide-gel electrophoresis. 2. Apparent Mr values of 31,900 for RhD polypeptide and 33,100 for Rhc,E polypeptide were obtained under both reducing and non-reducing conditions. Glycosylation of RhD,c and E polypeptides was not detected. 3. RhD,c and E immune complexes also contain a glycoprotein component. RhD glycoprotein (apparent Mr 45,000-100,000) is distinct from Rhc,E glycoprotein(s) (apparent Mr 35,000-65,000). Rh (Rhesus) glycoprotein carbohydrate moieties are susceptible to endo-beta-galactosidase digestion and carry blood-group-ABH determinants. This suggests the presence of polylactosaminoglycan-type structures. 4. Rh glycoproteins are not present in Rh immune complexes as a result of non-specific adsorption of membrane glycoproteins during the membrane-solubilization phase of immune-complex isolation because RhD immune complexes isolated from a 1:1 (v/v) mixture of Acde/cde and OcDE/cDE red cells do not contain blood-group-A-active glycoprotein. 5. Blood-group-A immune complexes isolated from group-A red cells of the appropriate Rh phenotypes contain the 31,900- and 33,100-apparent-Mr Rh polypeptides. 6. It was concluded from the above evidence that non-covalent Rh-glycoprotein-Rh-polypeptide complexes exist in the native red-cell membrane. 7. The 31,900- and 33,100-apparent-Mr Rh polypeptides are absent from blood-group-A immune complexes isolated from regulator type Rhnull cells (donor A.L.), but are replaced by a 33,800-apparent-Mr Rhnull-specific polypeptide (Rhnull polypeptide). It is suggested that Rhnull polypeptide is an aberrant product of the Rh gene complex.     

Assignment of the red cell antigen, Targett (Rh40), to the Rh blood group system.

Statistical and serological evidence from a large kindred and two unrelated adults indicates that Targett (Tar) is an antigen in the Rh blood group system and that its presence is assocciated with a weak expression of the Rh antigen D. In the numerical notation the Tar antigen is designated Rh40.     

Protein Kinase Activity in Hepatitis B Virus

Protein kinase activity was found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B virus-infected patients, in virion cores, and in hepatitis B core antigen particles purified from hepatitis B virus-infected hepatic tissue and was not found in purified hepatitis B surface antigen particle preparations free of Dane particles. Only a fraction of the major polypeptide (apparent size, 19,700 daltons) in Dane particle cores and hepatitis B core antigen particles from infected liver appeared to be phosphorylated, and phosphorylation changed the electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to that expected for a polypeptide of 20,600 daltons. Five minor polypeptides with apparent sizes between 38,000 and 63,000 daltons were phosphorylated in Dane particles and Dane particle core preparations but were not detected in hepatitis B core antigen particles from infected liver. None of these had electrophoretic mobilities corresponding to those of known hepatitis B surface antigen polypeptides. Prolonged storage of purified hepatitis B core antigen particles or incubation with human immunoglobulin G preparations containing antibody to the hepatitis B core antigen with or without antibody to the hepatitis B e antigen resulted in the conversion of the polypeptide with an apparent size of 20,600 daltons to ones with apparent sizes of 14,700 and approximately 6,000 daltons, suggesting proteolytic cleavage of the 20,600-dalton polypeptide under these conditions.     

High-frequency antigens of human erythrocyte membrane sialoglycoproteins, IV. Molecular properties of the U antigen

The nature of the common erythrocyte antigen U, that is absent from S-s-U-cells, which lack glycophorin B (Ss sialoglycoprotein), was investigated using six different antisera. The molecular features of a U-like antigen (Duclos), detected by a hitherto unique serum, were also studied. The U and Duclos antigens are complex in that they exhibit relationships with the MNSs and Rh blood group systems. Various fractionation, cleavage, or modification products of normal erythrocyte membranes were used in hemagglutination inhibition assays. Both, the U and Duclos antigens were found to represent labile structures that require lipids, at least for optimum expression of antigen activity. The antigens could be solubilized using conditions of Triton X-100 extraction that release glycophorin B, but solubilize the Rh antigens only to a small extent. Anti-U and anti-Duclos were also inhibited, albeit weakly, by glycophorin B-containing fractions obtained by chromatographic separation of Triton X-100 extracts. The residues approx. 33-39 of glycophorin B represent essential parts of the U antigen, as judged from proteolytic digestion and chemical modification. Conversely, the expression of Duclos activity seems to require a region of glycophorin B (C-terminal of the positions approx. 34-36) that could not be cleaved by various proteinases. Data obtained with anti-Duclos have to be interpreted with caution, since there is evidence that this serum might contain a mixture of antibodies.     

Biochemistry of the erythrocyte Rh polypeptides: a review

The clinically important Rh blood group system is complex, consisting of multiple distinct antigens. Despite clinical recognition for over 50 years, the Rh blood group antigens have remained poorly understood on a molecular level until the recent identification and characterization of the "Rh polypeptides," the core structural proteins of the Rh antigens. This group of erythrocyte membrane proteins of molecular weight 30,000-35,000 daltons was first recognized by employing Rh-specific antibodies to immunoprecipitate radiolabeled components of erythrocyte membranes. By using antibodies specific for the Rh D, c, and E antigens, a series of highly related non-identical proteins were immunoprecipitated, indicating that the Rh antigens are composed of multiple related proteins. The Rh polypeptides have been purified and characterized, and they were found to have several unusual biochemical characteristics. The Rh polypeptides penetrate the membrane bilayer; they are linked to the underlying membrane skeleton; they are covalently fatty acid acylated with palmitate. While the Rh antigenic reactivity is unique to human erythrocytes, the Rh polypeptides have been isolated from erythrocytes of diverse species and are thought to be fundamental components of all mammalian erythrocyte membranes. The functional role of the Rh polypeptides remains undefined, but a role in the organization of membrane phospholipid is suspected.     

Identification of cultured cell employing specific substance--with special references of "reserve cells" in uterine cervix

Identification of endocervical "reserve cell", which have been regarded as the origin of squamous cell carcinoma of the uterine cervix, was attempted employing immunohistochemically specific substances. The antigenicity of keratin, squamous cell carcinoma antigen(SCC), epithelial membrane antigen(EMA), tissue polypeptide antigen(TPA), vimentin, secretory component(SC) and placental alkaline phosphatase(PLAP) was investigated in histological preparations as well as cultured cells obtained from primary culture of endocervical tissue. The immunohistochemical findings in histological preparations revealed the following: a strongly positive reaction with TPA, a slightly positive reaction with EMA, a very slightly positive with SCC and PLAP, and a negative reaction with keratin, vimentin and SC. Cultured cells were divided into 4 groups according to their morphological characteristics; among these, small rounded or polygonal cells with a centric single nucleus showed similar immunocytochemical reactions to those of "reserve cells" in histological preparations, indicating that "reserve cell" can be growing in culture. The results obtained suggest that immunohistocytochemical specific substances may be useful to identify cultured cells.     

Evidence for a structurally homologous Rh-like polypeptide in Rhnull erythrocytes.

Human Rhnull red blood cells fail to react with Rh antibodies, indicating that these cells are either devoid of Rh protein or, like other species, possess antigenically distinct variants. To determine whether Rhnull cells possess an Rh-like polypeptide, 32-kDa proteins from D--, rr, and Rhnull cells were labeled with the cysteine-specific probe, 125I-labeled pyridyldithioethylamine. Size comparisons of labeled proteins in Triton X-100-solubilized membranes from Rh-bearing and Rhnull cells showed similar sedimentation coefficients and Stoke's radii. Immunoprecipitated Rh(D) from D-- cells, Rh(c) from rr cells, and purified 32-kDa proteins from Rhnull cells were digested with alpha-chymotrypsin and examined by high-performance liquid chromatography and by two-dimensional iodopeptide mapping. Analysis of 125I-labeled chymotryptic fragments from immunoprecipitated Rh(D) and Rh(c) showed the labeled peptides from both phenotypes to be virtually identical. High-performance liquid chromatography profiles and iodopeptide maps of 32-kDa Rhnull proteins yielded patterns identical to 32-kDa proteins isolated from D-- cells and rr cells with the exception of one missing 125I-labeled peptide. Further analysis of the Rh-related fragments from Rhnull cells showed significant homology with immunoprecipitated Rh(D) and Rh(c). DNA sequence analysis of cysteine-encoding regions from Rh-bearing and Rhnull cells showed complete identity. These data suggest that Rhnull red blood cells, although serologically distinct, possess an Rh-like protein that is structurally very similar to Rh(D) and Rh(c).