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At least 19 recordsLinked to original sources
PubMed1997

High-resolution solution NMR structure of the Z domain of staphylococcal protein A.

Staphylococcal protein A (SpA) is a cell-wall-bound pathogenicity factor from the bacterium Staphylococcus aureus. Because of their small size and immunoglobulin (IgG)-binding activities, domains of protein A are targets for protein engineering efforts and for the development of computational approaches for de novo protein folding. The NMR solution structure of an engineered IgG-binding domain of SpA, the Z domain (an analog of the B domain of SpA), has been determined by simulated annealing with restrained molecular dynamics on the basis of 671 conformational constraints. The Z domain contains three well-defined alpha-helices corresponding to polypeptide segments Lys7 to Leu17 (helix 1), Glu24 to Asp36 (helix 2), and Ser41 to Ala54 (helix 3). A family of ten conformers representing the solution structure of the Z domain was computed by simulated annealing of restrained molecular dynamics using the program CONGEN. The average of the root-mean-square deviations (r.m. s.d.) of the individual NMR conformers, relative to the mean coordinates, for the backbone atoms N, Calpha and C' of residues Phe5 through Ala56 is 0.69 A; the corresponding backbone r.m.s.d. for the three-helical core is 0.44 A. Helices 1, 2 and 3 are antiparallel in orientation (Omega12=-170(+/-4) degrees , Omega13=+16(+/-3) degrees , Omega23=+173(+/-7) degrees ). A comparison of backbone amide hydrogen/deuterium exchange rates in free and IgG-bound Z domains demonstrates that the amide protons of helices 1, 2 and 3 are protected from rapid exchange in both states, indicating that all three helices are also intact in the IgG-bound state. These solution NMR results differ from the previously determined X-ray structure of the similar SpA B domain in complex with the Fc fragment of a human IgG antibody, where helix 3 is not observed in the electron density map and from the solution NMR structure of the B domain, where helix 3 is observed but helix 1 is tilted by approximately 30 degrees with respect to helices 2 and 3. Hydrogen-bonded N-cap and C-cap formation is observed for all three helices of the Z domain; these capping interactions appear to be highly conserved in the five homologous domains of SpA.

Amino Acid Sequence↗
PubMed1995

VH3 family antibodies bind domain D of staphylococcal protein A.

Staphylococcal protein A (SpA) is a 45-kDa bacterial membrane protein that can interact with either Fc gamma, a constant region portion of IgG, or with the Fab portion that also mediates conventional Ag binding. In recent reports, SpA has been shown to specifically interact with Fab derived from the VH3 family and is little affected by VH CDR3, JH, or light chain usage. To identify a site on SpA responsible for VH3 Fab binding, we cloned and expressed in Escherichia coli the 61 amino acid sequence of SpA that represents domain D, and this small protein exhibited both the VH3 Fab and Fc gamma binding specificities. Surface plasmon resonance measurements demonstrated that domain D and native SpA had the strongest binding interactions with an IgM-kappa encoded by the germline configuration of the VH3 gene VH26c. In contrast, the apparent affinities for Fc gamma binding were at least fivefold weaker. A variant of domain D was also created that is devoid of the three-codon insertion that distinguishes domain D from all other domains in SpA. Although this deletion did not significantly affect the VH3 Fab-mediated SpA binding activity, it did improve the affinity of Fc gamma binding by an order of magnitude. These observations characterize a site on SpA responsible for binding interactions with B cell Ag receptors that are highly analogous to that of superantigens for T cell receptors.

Amino Acid Sequence↗
PubMed1994

Construction and characterization of M13 bacteriophages displaying functional IgG-binding domains of staphylococcal protein A.

Staphylococcal protein A (SPA) is ranked as a versatile probe in immunoassays because of its immunoglobulin G (IgG)-binding capability. However, poor binding of SPA to the IgG of some laboratory animals and its inability to bind human IgG3 restricts its universal utility. In the present study, DNA encoding the four IgG-binding domains of SPA (E, D, A and B) or the B domain alone has been fused, in separate phagemid vectors, to the 5' end of gene 111 of the phage M13. Upon infection by helper phage M13KO7, phagemid particles encapsulating single-stranded DNA were produced. Dot immunoblot and Western blot analyses showed the presence of fusion proteins on the M13 surface. Binding of rabbit IgG-horseradish peroxidase (IgG-HRP) complex to the phage particles confirmed that the fusion proteins possessed functional IgG-binding domains. The interaction of these phages with immobilised human IgG and its various subclasses was studied by the phage capture immunoassay where the captured phages were detected by a monoclonal antibody to the major coat protein encoded by gene VIII (gVIII). The phages showed maximal binding to IgG1 kappa, followed by IgG2 kappa, and showed negligible binding to the IgG3 kapa and IgG3 lambda subclasses. The specificity of IgG-binding phages was confirmed in a phage capture and elution assay where the binding of these phages to immobilised human IgG1 kapa weas abolished in the presence of excess of soluble protein A. Moreover, IgG-binding phages could be enriched approx. 1000-fold over non-specific phages in a single round of panning.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Induction of T cell growth factor synthesis in human peripheral blood lymphocytes by staphylococcal protein A.

Staphylococcal Protein A (SpA) induces T cell growth factor (TCGF) production in cultures of human peripheral blood mononuclear cells (PBMC) with titers equivalent to those induced by PHA. The optimal time and cell concentration for TCGF production were found to be the same for SpA and PHA . TCGF induction by SpA was blocked by addition of human AB-serum or human IgG, as was the mitogenic effect of SpA. An easy and inexpensive procedure is described for the quantitative removal of SpA from TCGF without loss of TCGF activity.

Binding Sites, Antibody↗
PubMed1998

In vivo inflammatory response to a prototypic B cell superantigen: elicitation of an Arthus reaction by staphylococcal protein A.

Staphylococcal protein A (SpA) is representative of a new class of Ags, the B cell superantigens (SAgs). These SAgs, unlike conventional Ags, bind to the Fab regions of Ig molecules outside their complementarity-determining regions. In addition, B cell SAgs can react with a substantial amount of a host's serum Igs by virtue of their ability to interact with many members of an entire variable heavy chain (VH) or variable light chain gene family. For example, SpA reacts with the Fabs of most human Igs using heavy chains from the VH3 gene family (VH3+). Members of this gene family are expressed on 30 to 60% of human peripheral B cells. We sought to determine whether the interaction of a B cell SAg with its reactive Igs can elicit immune complex-mediated tissue injury. Using the Arthus reaction in rabbits as an in vivo model of immune complex-mediated tissue inflammation, we demonstrated that untreated rabbits, which were administered SpA intradermally (i.d.), do not develop a cutaneous inflammatory response. However, when rabbits were pretreated i.v. with human IgG (hIgG), i.d. injections of SpA induced an inflammatory response with the classical histologic features of an Arthus reaction. To determine whether this Arthus-like response occurred via a B cell superantigenic mechanism, the rabbits were pretreated with VH3-depleted hIgG and then were administered SpA i.d. We found that the induction of a prominent inflammatory response by SpA was dependent upon the presence of VH3+ molecules in the hIgG pretreatment. These results provide compelling evidence that an interaction of the B cell SAg, SpA, with its reactive (VH3+) IgGs leads to an immune complex-mediated inflammatory response in vivo.

Animals↗
PubMed1975

Demonstration and assaying of IgG antibodies in tissues and on cells by labeled staphylococcal protein A.

Staphylococcal protein A (SpA) labeled with FITC was used to detect IgG antibodies to tissue and cell surface antigens. Radioiodinated SpA was employed in a radioimmuno technique to assay IgG antibodies which had reacted with cell membrane antigens. Red blood cells coated with SpA were used as an indicator system in the mixed haemadsorption technique to detect IgG antibodies on tissue culture cells. The results showed that SpA was highly specific in the various systems tested, although it was not as sensitive in the immunofluorescence test as FITC anti-IgG. It was confirmed that SpA reacts with IgG antibodies of human, monkey, rabbit, sheep and mice origin. The reactivity with human and monkey IgG was stronger than with IgG of the other species. We consider labeled SpA as an alternative to monospecific anti-IgG for detection and assaying of IgG antibodies to tissue and cell surface antigens.

Adrenal Glands↗
PubMed1986

Fusion of pro region of subtilisin to staphylococcal protein A and its secretion by Bacillus subtilis.

Subtilisin is synthesized as a preproenzyme in Bacillus subtilis. We fused that region of the subtilisin gene, (apr[BamP]), which encodes the signal sequence and pro region, to the mature gene sequence (spa) for a heterologous protein (staphylococcal protein A). B. subtilis cells harboring this gene fusion synthesized a fusion protein consisting of the signal and pro sequence of subtilisin fused to the protein A; the signal sequence was processed and a fusion protein (pro + protein A) was secreted into the growth medium.

Amino Acid Sequence↗
PubMed1994

Characterization of modified staphylococcal protein A containing phospholipid monolayer on both solution and slide surfaces.

A method is described for incorporation of water-soluble protein Staphylococcal protein A (SpA) into phospholipid monolayer using covalent protein-lipid conjugates in detergent solution. The amphiphilic conjugates have solubility properties very similar to intergral membrane proteins. When the conjugates are applied into dipalmitoyl-phosphatidic acid monolayer, a protein containing monolayer is formed on subphase surface. The monolayer is transferred to pre-coated substrate surface to form an artificial membrane. Results show that unmodified SpA is readily ejected from the monolayer when compressing the monolayer but modified SpA incorporates into the monolayer stably. The incorporation of the protein is proportional to the lipid coupling degree. When the protein is excessively modified, the IgG binding activity of the SpA in the membrane is lost significantly.

Immunoglobulin G↗
PubMed1992

Artificial immunoglobulin G-binding protein mimetic to staphylococcal protein A. Its production and application to affinity purification of immunoglobulin G.

Staphylococcal protein A consists of a single polypeptide with five immunoglobulin G (IgG)-binding domains, which are linked as E-D-A-B-C in this order from the amino terminal. The DNA coding domains A-B were polymerized one to six times linearly, taking advantage of the non-palindromic nucleotide sequence of the AccI recognition site and the resultant DNAs were inserted in pTRP vector carrying trp promoter. The artificial IgG-binding proteins [pA(AB)1-6], which had been expressed in Escherichia coli JM109, were purified by methods involving IgG-Sepharose affinity chromatography. Among pA(AB)1-6 immobilized on cyanogen bromide-Sepharose, pA(AB)4-Sepharose was the highest in IgG-binding capacity at the same level of mg protein per ml gel, about 30% higher than protein A-Sepharose. At 8 mg protein per ml gel, it bound and eluted about 24 mg of IgG from rabbit serum. Its IgG-binding capacities were the highest with porcine, rabbit, human and guinea pig sera, intermediate with bovine, horse and sheep sera and the lowest with mouse, goat, rat and chicken sera.

Amino Acid Sequence↗
PubMed1989

Dissociation between murine spleen cell mitogenic activity of enterotoxin contaminants and anti-tumor activity of staphylococcal protein A.

Soluble staphylococcal protein A (SpA) in the form of high m.w. complexes with IgG has been shown to significantly inhibit the growth of Meth A fibrosarcomas in BALB/c mice. Although SpA reportedly is a potent T cell mitogen that can induce immune cell proliferation and production of humoral factors with anti-tumor activity, it has been suggested that mitogenic enterotoxin contaminants might be responsible for these effects. The purpose of the present study was to investigate the nature of SpA-induced cell proliferation and the relationship between mitogenicity and the anti-tumor effect that we observed in our mouse model. SpA stimulated the proliferation of a mixed population of splenic B and T cells from BALB/c mice, but activity did not require the presence of IgG in the culture medium. Furthermore, mitogenic activity could be inhibited completely by anti-SEA plus anti-SEB, but was unaffected by anti-SpA. HPLC-purified SpA was inactive while the mitogenic factor(s) had the same retention time as authentic enterotoxin and its activity was inhibited by anti-SEA and anti-SEB, but not by anti-SpA. Enterotoxin-free rSpA produced in Escherichia coli had the same IgG binding capacity as the staphylococcal product but was not mitogenic. These data indicate that SEA and SEB completely account for mitogenicity in SpA preparations. In contrast, we found that optimal concentrations of rSpA as well as crude and HPLC purified staphylococcal SpA were equally effective in inhibiting the growth of established Meth A fibrosarcomas demonstrating that SpA is responsible for antitumor activity without any apparent role for enterotoxins.

Animals↗
PubMed1990

Interaction between heat shock protein DnaK and recombinant staphylococcal protein A.

When a protein derived from the immunoglobulin G (IgG)-binding domains of staphylococcal protein A was expressed in Escherichia coli and recovered from cell extract by IgG affinity chromatography, the 69-kilodalton heat shock protein DnaK was found to be copurified. DnaK could be selectively eluted from the IgG column by ATP or by lowering the pH to 4.7. Protein A could subsequently be eluted by lowering the pH to 3.2. Thus, this procedure allows a one-step purification of both DnaK and protein A from cell extract. In vitro experiments with pure DnaK and protein A revealed that DnaK did not interfere with the IgG-binding properties of protein A but associated with its unfolded C-terminal in a salt-resistant manner. In addition, a specific interaction between DnaK and denaturated casein was found.

Binding Sites↗
PubMed1995

Proteolysis of fusion proteins: stabilization and destabilization of staphylococcal protein A and Escherichia coli beta-galactosidase.

The product yield of staphylococcal Protein A reached only 1.8% of the cell dry weight, while the corresponding value was 14% for a fusion protein composed of Protein A and Escherichia coli beta-galactosidase [1], when produced in the same E. coli host strain, with the same promoter and under identical process conditions. Measurement of the stability of Protein A in vivo showed that it was quickly degraded in the cell with a half-life of 30 min when the protein was expressed alone, but after fusion to beta-galactosidase, the Protein A part became considerably stabilized. In spite of the fast intracellular proteolysis of Protein A, few degradation products could be identified on Coomassie Brilliant Blue-stained SDS/PAGE gels after IgG purification, indicating an even faster degradation of the Protein A fragments. Such degradation products, however, accumulated during incubation of the disintegrated cells. Intracellular degradation intermediates could be demonstrated with the more sensitive Western-blot technique. This technique also revealed that a slow degradation took place not only in the Protein A moiety of the fusion protein, but also in the beta-galactosidase moiety. A control with native beta-galactosidase also showed a weak in vivo proteolysis of this molecule, but it was more stable in free form than in the fused form. This means that the proteolytically very sensitive Protein A was stabilized by fusion with beta-galactosidase, but the originally rather stable beta-galactosidase became slightly more susceptible to proteolysis after the fusion.

Amino Acid Sequence↗
PubMed1986

Development of an enzyme-linked immunosorbent assay for staphylococcal protein A produced in Escherichia coli by pUC8 based plasmids containing the Staphylococcus aureus Cowan I protein A gene.

Staphylococcal protein A, a cell wall component of several strains of Staphylococcus aureus has found many uses as a research tool, as a diagnostic reagent and even as a possible therapeutic agent in cancer. These uses have arisen exclusively out of its almost unique property of binding specifically to the Fc region of many immunoglobulin molecules. As Staphylococcus aureus is pathogenic, it has been desirable for industrial purposes to clone the gene coding for protein A into Escherichia coli and to develop a sensitive assay free from interference by bacterial components. We describe an ELISA assay which is capable of detecting staphylococcal protein A in bacterial lysates at levels as low as 1.0 ng/ml and which is free of interference from lysozyme, lysostaphin, endogenous peroxidases or other bacterial antigens.

Animals↗
PubMed1987

Heterogeneity of binding of monoclonal IgA to staphylococcal protein A is related to the IgA polymerization state.

The human IgA2-lambda myeloma protein Fel consists of covalent dimers and monomers which are partially self-associated. Affinity chromatography of this protein on staphylococcal protein A-Sepharose revealed that approximately 8% of the protein was retained and eluted by acid buffer. Although retained protein Fel was highly aggregated, in the presence of dissociating agents mostly monomeric form was found. Affinity rechromatography and electrophoresis of both fractions from affinity chromatography revealed that the retained fraction possessed substantially higher affinity for SpA than did the nonretained one. This could be due to the multivalency of protein Fel aggregates.

Antibodies, Monoclonal↗
PubMed1991

The interaction of a protein from the coelomic fluid of earthworms with staphylococcal protein A.

A Staphylococcal protein A (SpA)-binding protein was isolated from coelomic fluids of the annelids, Lumbricus terrestris (LT) and Eisenia foetida (EF), by affinity chromatography on SpA-Sepharose. Analyses by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) under reducing and nonreducing conditions showed that SpA-binding activity is associated with a single chain protein, with a mol wt of 62 kD. The carbohydrate moiety of this protein consists of 21.4 g/100 g of O-linked and N-linked oligosaccharides. The amino acid composition of SpA-binding protein did not show structural homology with that of human IgG1 heavy chain (Daw), which also binds SpA.

Amino Acids↗
PubMed1988

A radioimmunohistochemical method for autoradiographic visualization of cell antigens using 125I-staphylococcal protein A.

125I-Staphylococcal protein A was used to visualize immunoreactive cell antigen in rat brain and pituitary by autoradiography. Autoradiograms of rat brain sections generated with 125I-protein A were clear and showed low background signals. We were able to visualize neural structures containing tyrosine hydroxylase or methionine-enkephalin-like immunoreactivities in the brain, and vasopressin-like immunoreactivity in the pituitary gland. Our results suggest that 125I-protein A can be used for the radioimmunohistochemical visualization of cell antigens in tissue sections.

Animals↗
PubMed1990

A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv.

A bifunctional molecule was genetically engineered which contained an amino-terminal effector domain that bound immunoglobulin Fc (fragment B of staphylococcal protein A) and a carboxyl-terminal domain that bound digoxin [a single-chain Fv (sFv)]. Effector and sFv binding properties were virtually identical with those of the parent molecules, despite the proximity of the FB to the sFv combining site. This finding is unprecedented since in all molecules of the natural immunoglobulin superfamily, the antigen binding domain is amino terminal to the effector domain. The FB-sFv sequence was encoded in a single synthetic gene and expressed as a 33,106 molecular weight protein in Escherichia coli. After purification, renaturation, and affinity isolation, yield of active fusion protein were 110 mg/L of fermented cells (18.5-g cell paste). Bifunctionality was confirmed by the ability of FB-sFv to cross-link IgG to digoxin-bovine serum albumin, as measured by plate assays and by Ouchterlony analysis. Analysis of the expressed fusion protein suggests that the sFv holds promise for the development of multifunctional, targetable single-chain proteins.

Amino Acid Sequence↗