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S Bhakdi

Publications and source records attributed to S Bhakdi.

16 recordsLinked to original sources
PubMed1992

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.

Actins↗
PubMed1979

Evidence for a two-domain structure of the terminal membrane C5b-9 complex of human complement.

Lipid vesicles carrying the purified membrane C5b-9 complex [C5b-9(m)] of complement were analyzed immunochemically and in the electron microscope after treatment with a combination of trypsin and alpha-chymotrypsin. Under reducing conditions, the externally oriented annulus was removed. The remaining part of the C5b-9(m), representing approximately half of the total mass of the macromolecular complex, was visualized in the electron microscope as a hollow cylindrical structure with walls of 1-nm thickness. This structure remained tenaciously attached to the lipid bilayer, projecting 8-9 nm from the external membrane surface into the aqueous environment. Cleavage of C5b-9(m) by proteolysis and reduction resulted in a sharp reduction of tis antigenic determinants. One hydrophilic protease-resistant C5 derivative was released from the membrane and recovered in the fluid phase. The membrane-bound residue almost totally lacked antigens precipitable with antisera to C5, C6, C9, and C5b-9(m).

Complement C5↗
PubMed1979

Proteolytic transformation of SC5b-9 into an amphiphilic macromolecule resembling the C5b-9 membrane attack complex of complement.

Proteolysis of fluid-phase SC5b-9 left a major part of the macromolecule intact and caused transition of the molecule from a hydrophilic to an amphiphilic state. The transformed complex exhibited neoantigens characteristic of the C5b-9 membrane attack complex of the complement. It yielded an SDS gel electrophoresis pattern that was similar, but not identical to that of the proteolysed, membrane attack complex. The proteolytically altered SC5b-9 complex bound lipid and incorporated into artificial lipid vesicles to yield a membrane-bound structure resembling the C5b-9 complement lesion.

Antigens↗
PubMed1978

Molecular nature of the complement lesion.

The principle molecular event leading to membrane perturbation by complement is the assembly of the terminal five serum complement components (C5b-C9) into a macromolecular C5b-9 complex on the target membrane [Müller-Eberhard, H.-J. (1975) Ann. Rev. Biochem. 44, 697--723]. The present communication reports on the ability of purified C5b-9 complexes isolated from target membranes to become reincorporated into artificial lipid vesicles. The data indicate that the complex is a vertically oriented, hollow, cylindrical macromolecule possessing lipid-binding regions that enable one terminus to penetrate into the lipid bilayer. A transmembrane pore appears to be created at the attachment site of the C5b-9 complex.

Animals↗
PubMed1978

Complement lysis: the ultrastructure and orientation of the C5b-9 complex on target sheep erythrocyte membranes.

The C5b-9 complex derived from human serum and assembled on target sheep erythrocyte membranes is a thin-walled cylinder rimmed by an annulus at one end. The total height of the cylinder is 150 A, towards which the annulus contributes 30 A. The cylinder has an apparently uniform internal diameter of 100 A. The external diameter of the annulus is 200 A. The classical complement 'rings' visualized on membranes after complement lysis represent such C5b-9 cylinders perpendicularly oriented on the membranes. The thin-walled cylinder is anchored in the membrane matrix and the annulus located in the exterior membrane glycocalyx. At the sites of attachment of the C5b-9 complexes, the continuity of the membrane bilayer is disturbed and the presence of trans-membrane pores is indicated. The data essentially support the 'doughnut' theory of complement lysis.

Animals↗
PubMed1978

Complement lysis: evidence for an amphiphilic nature of the terminal membrane C5b-9 complex of human complement.

The terminal, membrane-derived C5b-9 complex of human complement (C) is an apparently hollow, cylindrical macromolecule vertically oriented on the target membrane. In the present study, an antiserum to the complex has been used to probe its immunobiochemical properties. "Neoantigenic" determinants characteristic of the complex have been detected, which are absent on native C5-C9 molecules. Evidence that the C5b-9 complex is an amphiphilic molecule that possesses apolar, detergent-binding surfaces has been obtained by using charge-shift crossed immunoelectrophoresis, and by direct demonstration of Triton X-100 binding to the complex in quantitative immunoelectrophoresis. By the same criteria, serum C5, C6, and C9 are hydrophilic molecules. The results indicate that assembly of C5-C9 into the terminal membrane C5b-9 complex is accompanied by conformational changes in the individual C components that lead to the exposure of apolar molecular regions in the complex. It is proposed that this constitutes the basis for the lipid-binding properties of the macromolecule, which enable it to become inserted into biologic and artificial lipid membranes with apparent generation of a transmembrane pore.

Animals↗
PubMed1977

Detection of amphiphilic proteins and peptides in complex mixtures. Charge-shift crossed immunoelectrophoresis and two-dimensional charge-shift electrophoresis.

Charge-shift electrophoresis has been suggested as a simple and novel method for differentiating between emphiphilic and hydrophilic proteins (Helenius, A. and Simons, K. (1977) Proc. Natl. Acad. Sci. U.S. 74, 529-532.) This communication reports on the combination of charge-shift electrophoresis with second dimensional quantitative immunoelectrophoresis, and on a two-dimensional modification of the charge-shift electrophoresis technique. From results obtained with unfractionated human plasma proteins and human erythrocyte membrane proteins we conclude that these modifications reliably permit detection of amphiphilic proteins and peptides in complex mixtures.

Blood Proteins↗
PubMed1976

The major "intrinsic" membrane protein of human erythrocytes. Preparative isolation and immunoelectrophoretic analyses.

(1) Preparative dodecyl sulfate gel electrophoresis of human erythrocyte membrane proteins has been used to isolate dodecylsulfate band 3 containing the M,N-glycoprotein and the major "intrinsic" membrane protein (Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617; Bretscher, M.S. (1971) J. Mol. Biol. 59,351-357; Bretscher, M.S. (1971) Nat. New Biol. 231, 229-232 and Marchesi, V.T. and Andrews, E.P. (1972) Science 174, 1247-1248). Subsequent isoelectric focusing in polyacrylamide gels containing Triton X-100 separates these two entities and allows their simultaneous purification. (2) The proteins thus obtained retain their antigenic properties. They are pure according to electrophoretic and immunoelectrophoretic criteria. However, crossed immunoelectrophoresis yields evidence for molecular microheterogeneity of the major "intrinsic" protein. (3) Analyses utilizing crossed immunoelectrophoresis with antibodies absorbed with intact erythrocytes show that the major "intrinsic" protein possesses antigenic determinants on both membrane surfaces and therefore spans the erythrocyte membrane. All determinants of the M,N-glycoprotein detectable with our antibodies were found solely on the exterior membrane surface. (4) Neither the major "intrinsic" membrane protein nor the major M,N-glycoprotein bound significantly to concanavalin A in crossed immunoaffinoelectrophoresis.

Amino Acids↗
PubMed1976

Isolation of the terminal complement complex from target sheep erythrocyte membranes.

(1) Membranes from sheep erythrocytes lysed with antibody and human complement were solubilized in Triton X-100 and subjected to isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Membrane-bound serum proteins were located in the gels by subsequent immunoelectrophoresis against antisera to human serum proteins. Monospecific antisera against C9 and C5 were used to locate the terminal complement complex, which is not dissociated by Triton X-100. The complex focused between pH 5.8 and pH 6.5 and was separated from the bulk of other membrane-bound serum proteins, which focused at pH ranges below than 6.0. (2) In a second step, proteins electrophoretically eluted from the gel sections containing the terminal complement complex were chromatographed on Sepharose 6B equilibrated with 0.05% Triton X-100. Fused rocket immunoelectrophoresis was used to monitor separations. This step separated the terminal complement complex from the remaining contaminating proteins. The complex eluted in a broad peak corresponding to a molecular weight range of 800000-4000000. (3) The terminal complement complex thus obtained migrated with alpha-mobility and yielded a single precipitation arc in crossed immunoelectrophoresis using polyvalent antisera to human serum proteins. A distinct precipitate was obtained with monospecific anti-C9. The presence of C5 and C6, in complex with one another and with C9 was demonstrable by immuno-double-diffusion. No immunoprecipitate was obtained with antisera to sheep erythrocyte membrane proteins. (4) Dodecyl sulfate gel electrophoresis of the complex revealed seven protein bands of 190000, 160000, 115000, 93000, 85000, 68000 and 60000 daltons. Planimetric quantitation of densitometric scans gave a molar ratio of approx. 0.7:0.3:1:1:1:2:1 for these bands, respectively. All bands stained faintly with periodate-Schiff. Two-dimensional dodecyl sulfate gel electrophoresis showed that the first two bands (190000 and 160000 daltons, probably C5b and C5c) represented proteins possessing more than one peptide chain linked by disulfide bonds. The main subunit for both bands was a protein of approximately 68000 daltons. Band 5 (83000 daltons, probably C8alpha) was split into two peptide chains of approximately 68000 and 15000 daltons. The other components were not affected by dithiothreitol treatment. (5) The dodecyl sulfate gel electrophoretograms obtained were very similar to that described by Kolb and Müller-Eberhard (Kolb, W.P. and Müller-Eberhard, H.J. (1975) J. Exp. Med. 141, 724-735) for the terminal complement complex isolated from inulin-activated serum. However, certain minor but consistent deviations were observed. A preliminary correction of the electrophoretograms is presented.

Animals↗
PubMed1975

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.

Antibody Specificity↗
PubMed1975

Immunochemical analyses of membrane-bound complement. Detection of the terminal complement complex and its similarity to "intrinsic" erythrocyte membrane proteins.

(1) Membranes of sheep erythrocytes lysed with antibody and human or rabbit complement were solubilized in non-ionic detergents (Triton X-100 or Berol EMU-043) and analysed immunochemically using antisera directed against individual complement components. The precipitation behaviour of membrane-bound C3, C5, C6 and C9 components of complement was examined by immuno-double diffusion, rocket- and crossed immunoelectrophoresis performed in agarose gels containing 1% non-ionic detergent. (2) Membrane-bound C5, C6 and C9 are antigenically altered compared with the native (serum) components. (3) Immuno-double diffusion in the presence of non-ionic detergents reveals formation of C5-C6-C9 complexes on the membranes; these complexes are stable in non-ionic detergent. No complex formation was detected in serum between native C5, C6 and C9 components. There was also no evidence for complexing between membrane-bound C3, C4 or membrane proteins and the "late-reacting" complement components. (4) The extractability of complement components by various manipulations has been studied by use of quantitative rocket immunoelectrophoresis. Up to 65% of membrane-bound C3 is readily extracted by dialysis of membranes against 1mM EDTA, pH 8.0, 100 mM EDTA, pH 8.0, 1.2 NaCl plus or minus EDTA, by extraction in isotonic buffers at 37 degrees C, by heating at 45 degrees C over several hours, or by treating membranes with 1 mM p-chloromercuribenzoate sulfonate. In contrast, less than 6% of the terminal complement complex can be eluted by any of the described methods or combination of methods. (5) Our data suggest that the terminal complement complex associates with membrane "core" components through apolar interactions.

Animals↗
PubMed1975

Two-dimensional separation of erythrocyte membrane proteins.

1). Erythrocyte membrane proteins eluted with Triton X-100 or dilute EDTA have been separated two-dimensionally by isoelectric focusing in polyacrylamide gels containing 1 percent Triton X-100 plus 8 M urea, followed by electrophoresis using sodium dodecyl sulfate. Characteristic patterns, consistent among 40 healthy donors, were obtained. 2. The resulting patterns contain at least 30 components. The "spectrin" components (sodium dodecyl sulfate Bands 1 and 2) focus in the same pH range. Other membrane components giving single bands in sodium dodecyl sulfate electrophoresis appear to be heterogeneous. 3. Triton X-100, but not EDTA, extracts the principal membrane glycoproteins and the major "intrinsic" protein. Otherwise, proteins preferentially eluted by EDTA extract poorly with Triton X-100 and vice versa. 4. Membrane glycoproteins migrate anodally during electrofocusing and can be purified in a simple, one-step procedure.

Cell Membrane↗
PubMed1975

Rapid preparative isolation of major erythrocyte membrane proteins using polyacrylamide gel electrophoresis in sodium dodecylsulfate.

1. We describe a simple method for preparative, sodium dodecylsulfate/polyacrylamide gel electrophoresis of the major proteins in human erythrocyte membranes. 2. The method is based on extraction of prestained proteins from gel slabs. Three different fluorescent dyes (o-phthalaldehyde, fluorescamine and 1-dimethyl-aminonaphthalene-5-sulfonylchloride) have been used for pretaining. The method allows separation of up to 75 mg membrane protein and isolation of mg quantities of all major erythrocyte ghost proteins, while preserving the high resolution of analytical polyacrylamide gel electrophoresis. 3. Yield depends on extraction conditions and the molecular weight of the proteins being eluted. It ranges from 43-48% for protein 1 (apparent mol. wt approx. 310000) and 72-78% for protein 3(apparent mol. wt 87 000-93 000) to 87-93% for protein 6 (apparent mol. wt 35 000). 4. The labile behaviour of the high molecular "spectrin" bands (bands 1 and 2) is described. Extraction at room temperature tends to split these proteins into products of lower molecular weight. In contrast, the minor protein components 2.1 and 2.2 tend to aggregate yielding components 1 and 2. 5. N-terminal amino acid analyses have been performed on proteins 1, 2, 3, 4A, 4B, 5 and 6. Each of these bands contains several N-terminals, most of which appear constant. Some additional N-terminal amino acids vary from one donor to the next.

Amino Acid Sequence↗