Search

Adsorption chromatography of streptococcal extracts on immobilized fibrinogen allows isolation of components that are linked to the corresponding receptors. In this study it is shown by an indirect bactericidal test that fibrinogen binds the M proteins of the streptococcal strains used. Phage-associated lysin extracts of group A type 1 streptococci precipitated with fibrinogen in a double-diffusion test. Fibrinogen reactive components of other streptococcal types inhibited this precipitation reaction. This suggests that the fibrinogen receptors in different types of group A streptococci have identical activity. The interaction between M protein and fibrinogen does not interfere with the interaction between M protein and the corresponding type specific antibodies. The streptococcal antigen components isolated by immobilized fibrinogen showed mitogenic activity in a lymphocyte transformation test.

The isolated 35 kDa fragment of protein G obtained by papain digestion of group G streptococci was found to bind solid phase intact IgG, Fc (2C gamma 2 + 2C gamma 3 domains), F(ab')2 and F(acb)2 (F(ab')2 + 2C gamma 2 domains) fragments but not pFc' (2C gamma 3 domains) fragments. The level of binding to rabbit F(acb)2 and rabbit F(ab')2 fragments was similar. Protein G binding to solid phase Fc fragments was inhibited by IgG, Fc, staphylococcal protein A and its monovalent fragment D, but was enhanced by F(ab')2 fragments. Chemical modification of tyrosine but not histidine residues of IgG abrogated its ability to inhibit the binding of protein G to solid phase Fc fragments. Protein G was found to strongly inhibit the binding of a monoclonal and a polyclonal human rheumatoid factor to IgG. These findings indicate that protein G binds with separate sites to the Fc and F(ab')2 fragments of IgG, that the interaction with the Fc fragment occurs at the C gamma 2-C gamma 3 domain interface region and that tyrosine but not histidine residues in this area are likely involved. The relationship of the Fc fragment-binding site specificity of protein G to that of other microbial IgG binding proteins and human rheumatoid factors is discussed.

A cell separation technique was designed based on the interaction between cell-surface-bound IgG and protein A of Staphylococcus aureus . The density of lymphoid cells coated with IgG antibodies against one of the surface markers was increased by adherence of staphylococci. Cells with adhering bacteria were separated from cells without bacteria by density gradient centrifugation in 11.5% sodium metrizoate. Bacteria were removed from the lymphoid cells by lysostaphin digestion. The purity of separated cells was approximately 95% even when the proportion of a specific cell population was below 10% in the initial mixture. The viability and the ability of cells to multiply in vitro were not significantly impaired by the fractionating procedure. The technique can generally be applied for cell separation, provided antibodies of the IgG class against specific surface markers are available.

Work from our laboratories has shown that the major antigenic determinants for rheumatoid factors (RFs) are in the C gamma 2-C gamma 3 interface region of IgG in the same area that binds staphylococcal protein A (SPA). Furthermore, the Fc binding proteins of groups A, C and G streptococci as well as the Fc binding proteins induced on cell surfaces by herpes simplex virus type I also bind to the same area of IgG. These binding site similarities between RFs and the microbial Fc binding proteins suggested conformational similarities between the RF antigen combining regions and the Fc binding regions of the microbial proteins. This hypothesis was supported by the observation that antibodies to SPA bind to the antigen combining regions of most RFs as well as to the Fc binding region of the T15 group A streptococcal Fc binding protein. These findings indicate that RFs bear the conformational internal image of these microbial proteins and suggest that RFs could arise as antibodies to the idiotypic determinants on antibodies to microbial Fc binding proteins. Alternatively, microbial Fc binding proteins could present IgG to the immune system in a way that renders specific areas of the C gamma 2-C gamma 3 interface region immunogenic. These relationships between RFs and microbial Fc binding proteins may prove to be important for our understanding of the generation of RFs in rheumatoid arthritis.

Human C3d (try-C3d), prepared from trypsin-digested C3, was fragmented by cleavage with CNBr. Eight peptides were defined and separated by h.p.l.c. on reversed-phase columns. By automatic Edman degradation the complete sequences of five peptides and partial sequences of three peptides were determined. To obtain overlapping peptides the latter three fragments were digested with trypsin, chymotrypsin or Staphylococcus aureus V8 proteinase, after which the fragments were separated on reversed-phase columns. Two of the CNBr-cleavage peptides were completely sequenced, and 70% of the sequence of the remaining CNBr-cleavage peptide was determined. The non-sequenced part represents a very hydrophobic segment of try-C3d. The sequence data obtained represent 90% of the primary structure of try-C3d. Alignment of the CNBr-cleavage fragments was made easier by comparison with the cDNA sequence of mouse pro-C3 [Wetsel, Lundwall, Davidson, Gibson, Tack & Fey (1984) J. Biol. Chem. 259, 13857-13862]. Comparison of try-C3d with the equivalent part of human C4B revealed an extensive sequence homology in the N-terminal half of the molecules.

Previous work has shown that IgG rheumatoid factors (RF) bind to the C gamma 2-C gamma 3 interface region of human IgG in the same area that binds staphylococcal protein A (SPA). Group A, C, and G strains of Streptococci possess Fc receptors that bind to IgG but not to fragments containing only the C gamma 2 or C gamma 3 domains. This work describes the binding site location on human IgG for the binding of the isolated Fc receptor from the T15 strain of a Group A streptococcus and its relationship to the site that binds SPA and the IgG RF. The isolated T15 Fc receptor (T15) with a molecular mass of 29.5 kD inhibited the binding of IgG RF to IgG. The binding of T15 itself to IgG was strongly inhibited by SPA (42.0 kD) and its monovalent fragment D (7 kD). Human IgG fragments consisting of the C gamma 3 domains did not inhibit the binding of T15 to IgG, whereas those with both domains were effective inhibitors. T15 did not bind to rabbit IgG fragments consisting of either the C gamma 2 or C gamma 3 domains, but did bind to those with both domains. An IgG3 myeloma protein was a poor inhibitor and has been shown to bind poorly to the IgG RF. Most IgG3 myeloma proteins did not bind to SPA. The substitution of Arg and Phe for His 435 and Tyr 436 is responsible for the poor binding of IgG3 to SPA and to the IgG RF. Chemical modification of His or Tyr on IgG reduced its ability to inhibit the binding of T15 to IgG. Reversal of the chemical modifications with hydroxylamine resulted in near complete restoration of inhibitory capacity. This information, collectively, coupled with the known positions in space of the His and Tyr residues in the C gamma 2-C gamma 3 interface region, verified that both His 435 and Tyr 436, and possibly His 310 and 433, are involved. These residues are also involved in binding SPA and the IgG RF. These data therefore indicate that the T15 Group A Streptococcal Fc receptor binds to the same location on the Fc of IgG as SPA and the IgG RF. The biologic relevance of these similarities between bacterial cell wall Fc receptors and IgG RF are not yet apparent, but suggest that RF could bear the internal image of these bacterial structures.

Preparations of protein A (SpA) from Staphylococcus aureus and low-molecular-weight SpA induce production of interferon-gamma (IFN-gamma) and are potent mitogens when added to human lymphocytes. The IFN-gamma-inducing and main mitogenic activity of these preparations can be separated from SpA by gel filtration and affinity chromatography. These activities can also be partially inhibited by antiserum to staphylococcal enterotoxin A (SEA) in a specific manner. It is concluded that the IFN-gamma-inducing activity and most of the mitogenic activity of SpA preparations are not attributable to intact or low-molecular-weight fragments of SpA but depend on the presence of SEA and other non-SpA products in the preparations of SpA.

M proteins of Streptococcus pyogenes types 1 and 12 were purified by immunochromatography on immobilized type-specific opsonizing antibodies. The M proteins were characterized serologically and biochemically. They absorb after immobilization opsonizing antibodies and only precipitate with homologous antisera in immunodiffusion. In SDS PAGE they show one main band, corresponding to mol. weights of 5 × 10 4 (type 1) and 5.4 × 10 4 (type 12), accompanied by faint lines, which form with the main band a precipitation line of identity in SDS-crossed immunoelectrophoresis. Both M proteins aggregate blood platelets and clot fibrinogen.

Various preparations of Staphylococci protein A (SpA) obtained either from strain Cowan-1 or strain A676 and two SpA low molecular fragments (AB and B) were tested for their mitogenic and NK-stimulating activity on human peripheral blood lymphocytes. Though all 4 preparations of SpA and both of its fragments were able to react with human IgG, apparently with both Fab and Fc sites of IgG, only the SpA preparations derived from A676 strain triggered the proliferation and potentiated the NK-activity of treated cells. The parallelism between the mitogenic and NK-stimulating activities of SpA suggests that both activities might be under the control of the same portion of the SpA molecule located in the non-immunoglobulin binding region of the molecule.

Crystals of the complex formed by human Fc fragment and fragment B (FB) of protein A from Staphylococcus aureus were prepared and the crystal structure determined at high resolution by multiple isomorphous replacement. Phase were improved considerably by combining these phases with calculated phases from the Fc component. FB is a small globular protein built of three parallel helices arranged in a triangular array. It binds by the first two helices of Fc and is attached to segments of CH2 and CH3. The CH3 module is unchanged between complex and Fc fragment crystals, but CH2 changes its position slightly relative to CH3. In addition, the upper third of CH2 is disordered in the complex crystals. Possible sources of this disorder are discussed.

Protein A from Staphylococcus aureus added to guinea pig serum inactivates the hemolytic complement (C′) of the serum. Preformed aggregates of protein A and guinea pig or human IgG inactivate complement. It is probable that the complement-consuming property of protein A is due to its ability to combine with the Fc part of the IgG, thus rendering the IgG complement-fixing. The possibility that the in vivo sensitivity-promoting activity of protein A is due to its ability to activate the complement system is discussed. A hemagglutination technique was developed in order to determine small amounts of protein A.

Summary Cutaneous and systemic anaphylaxis-like reactions as well as hemorrhagic reactions were produced in nonimmunized guinea pigs by injection of a bacterial cell wall protein (protein A) from Staphylococcus aureus. These phenomena are believed to be mediated by a reaction between protein A and the Fc region of γ-globulin. Protein A is thus able to produce in normal guinea pigs reactions indistinguishable from immediate hypersensitivity reactions.

Summary Arthus-like reactions in rabbits are produced by the interaction of human γG-globulin and a cell wall protein (protein A) from Staphylococcus aureus. The skin lesions are produced either by intradermal injection of preformed protein A-γ-globulin precipitates or by intradermal injection of protein A into rabbits that had previously received human γG-globulin intravenously. The skin reaction is regarded as not due to an antigen-antibody reaction but rather to a pseudo-immune reaction between protein A and human γG-globulin.

THIS X-ray Crystallographic study of a valine specific transfer RNA (tRNAval) from yeast demonstrates that the degree of order obtainable in crystals of the tRNA is sufficient to give sharp diffraction spots to a resolution of 3 A. This degree of resolution is comparable with that from crystalline globular proteins and therefore the determination of the structure of this tRNA at atomic resolution should be possible. A detailed account of the crystallization procedure is given.

When normal human or mouse serum is added to micro ELISA plates coated with monomeric or aggregated IgG, complement component C5 binds to IgG. C5 binding was demonstrated with a specific chicken anti-C5 antibody. Hydrazine treatment of the serum or addition of EDTA to the serum abolished the binding of C5. C5-deficient mouse serum was negative for C5 binding, whereas the same serum supplemented with human C5 restored the binding of C5. Chicken anti-C5-coated plates were used for determination of C5-containing circulating immune complexes (CIC). Increased concentrations of CIC were found in sera from patients with rheumatoid arthritis and Bell's palsy.

Extracorporeal immunoadsorption with protein A (SpA) containing Staphylococcus aureus Cowan I (SAC) has previously been shown to induce an antitumor and antiviral response in some feline leukemia virus (FeLV)-infected, lymphosarcoma (LSA) cats. However, the mechanism by which this response is induced is unknown. Since it is possible that SpA dissociates from the SAC column during treatment, and that intravenous infusion of SpA could be a more efficacious form of treatment than extracorporeal immunoadsorption therapy, 6 normal cats and 15 FeLV-infected pet cats with naturally occurring leukemia or LSA were infused with SpA. No toxic effects resulting from SpA infusion were observed in the normal cats. Antitumor effects were observed in one of the eight cats with LSA and in three of the six cats with leukemia, but there were no antiviral responses. The antitumor responses were objective regressions; however, all tumors recurred. These results demonstrate that infusion of SpA into FeLV-infected LSA or leukemic cats can result in an antitumor but apparently not in an antiviral response.

Mouse macrophages and human peripheral blood lymphocytes exhibiting on the membrane autologous IgG as well as mouse thymocytes coated with human IgG, showed a strong cell concentration dependence of surface IgG disappearance during an incubation at 37 degrees C. Mouse spleen lymphocytes treated with anti-mouse Ig behaved in the same way regarding the disappearance of surface immunoglobulins, while the redistribution of ligands (cap formation) was not dependent on cell concentration.

A direct method for demonstrating antigenic markers on cell surface has been developed which makes use of the ability of bovine red blood cells coated with protein A of Staphylococcus aureus (ES) to binding IgG antibody without agglutinating. Red cell-linked rabbit IgG antibody reagents (ESA) were prepared in this way, and Ig-, IgM- and Thy-1-bearing mouse spleen and peripheral blood lymphocytes were identified by a rosette assay.

The effect of protein A of Staphylococcus aureus (SpA) on the binding of rabbit IgG to the Fc receptors of mouse lymphocytes and macrophages was found to correlate with the aggregation of the IgG ligands. After binding anti-erythrocyte IgG complexed with SpA, the cells were able to attach to and kill erythrocyte indicator cells with a higher efficiency than lymphoid cells treated with anti-erythrocyte IgG alone. The amount of anti-peroxidase IgG which can be bound to effector cells was not changed by reaction with SpA. In contrast, the binding to cells of IgG-coated erythrocytes and of anti-peroxidase IgG complexed with peroxidase was substantially reduced by reaction with SpA. The results are compatible with the presence of two distinct Fc receptors, one for cytophilic monomeric IgG and another for polymeric (antigen-complexed) IgG.

Fluorescent protein A of Staphylococcus aureus (F1-SpA) acts on the IgG receptors of guinea-pig lymphocytes (GPL) and human lymphoid cells (Seraphina) to induce a redistribution pattern (multiple spots, patches, expelled material, caps) similar to the membrane staining of some living cells with anti-IgG. No ring staining was observed, which implies that F1-SpA acts as a multivalent cross-linking agent on IgG receptors. A prozone effect dependent on F1-SpA concentration was observed.

The binding site of immunoglobulin G (IgG) to herpes simplex virus (HSV) type 1-induced Fc receptor was investigated using human IgG Fc intermediate (Fc(i)) fragments, fragment D of staphylococcal protein A (SPA) and chemically modified human IgG. Human IgG Fc(i) fragment composed of one Cgamma2 and two Cgamma3 domains, bound strongly to HSV-1-infected cells. Fragment D, a monovalent subunit of SPA, inhibited the binding of radiolabelled human IgG Fc fragments to the HSV Fc receptor. Reductively methylated human IgG reacted equally well to HSV-infected cells, as did chemically unmodified IgG in contrast to N-acetylimidazole-modified and diethylpyrocarbonate-modifed human IgG, which were unreactive. These results suggest a similar binding site on human IgG for SPA and the HSV-1 Fc receptor with involvement of the amino acid residues Tyr and His but not Lys. The similarities of binding sites on the IgG molecule for the HSV-1 Fc receptor and rheumatoid factors (RF) may be important for understanding the mechanism of RF production in rheumatoid arthritis or other disease states.

A latex agglutination assay for the detection of protein A-secreting Staphylococcus aureus strains or strains with protein A in the cell wall is described. The assay utilizes latex particles coated with chicken anti-protein A antibodies. Chicken antibodies do not react with protein G-producing streptococci or rheumatoid factor, thus avoiding false-positive reactions.

The affinity and stoichiometry of interaction between staphylococcal protein A and different domains of immunoglobulins have been studied. Light scattering and tryptophan fluorescence quenching titrations along with direct binding assays were performed. The lack of binding to protein A of pFc′ fragment (corresponding to CH3 domain of IgG) or of Facb derivative of rabbit IgG (which is devoid of the CH3) suggests that the locus of protein A binding is at the interface between the CH2 and CH3 domains. This assignment is also supported by results of the tryptophan fluorescence quenching and C 1 binding experiments.

The relation between the M-component (`paraprotein') and the maximal antistreptolysin-O (AST) activity is studied in the sera from two patients, one with unequivocal and one with probable myeloma. The results indicate that the AST activity is to be found in the M-components and not in the `normal', diffuse γ-globulin.