Sandbox 300

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Contents

Introduction

PDB ID 1ss1

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Staphylococcal protein A
Gene: SPA (Staphylococcus aureus)
Resources: FirstGlance, OCA, RCSB, PDBsum
Coordinates: save as pdb, mmCIF, xml



Staphylococcal protein A is originally a component of the cell wall in over 90% of Staphylococcus aureus strains[1]. This protein, which is also called immunoglobulin G binding protein A is covalently linked to the peptidoglycan of the cell wall[2]. Nowadays the protein is often used for biochemical analysis due to its structural and biochemical properties. The expression of the chromosomal spa gene, which encodes the protein A, is regulated by the agr system and Rot. The level of expression is up-regulated by Rot and down-regulated by agr. Furthermore protein A serves as a virulence factor to Staphylococcus aureus . It is essential for colonization and infections mediated by this kind of bacteria[3].


Structure of the protein

The structural details of protein A were solved by the nuclear magnetic resonance method. The length of the amino acid chain of protein A contains 508 residues. The amino acids cystein and tryptophan do not occur in the amino acid sequence. The molecular weight of the described protein is 55439 Dalton and it consists of only one protein chain. The 3D structure[4] is build up of three α-helixes and it consists of five extracellular domains, which are designated as E, D, A, B and C. Furthermore the protein contains cell-wall spanning regions, called Xr and Xc, and a hydrophobic membrane spanning domain, which is distal to LPXTG and consists of 18-20 residues[5]. Protein A exists in a secreted and a cell wall anchored form. If it is bound to the cell wall of Staphylococcus aureus it is covalently linked to the peptidoglycan via its C-terminal domain.

Image:Spa-1.jpg


Function associated to this protein

Protein A has been identified as a cell surface protein of Staphylococcus aureus which contributes to the staphylococcal virulence. The virulence is mediated by the ability to interact with plasma proteins[6]. Protein A is able to bind to the Fc (constant region of IgG which is involved in effector functions) and the Fab fragment (also a part of Ig which is responsible for antigen recognition). The interactions of protein A with plasma proteins are mediated by five homologous domains, which are called E, D, A, B and C. Each of the homologous repeated domains comprises 56-61 residues, which are followed by a polymorphic variable repeated region, which is called Xr, and a conserved region Xc, which includes a cell wall attachment sequence[7]. Each domain is able to bind one IgG molecule through its Fcγ binding site . The binding of protein A to the Fc fragment plays a major role in the virulence of Staphylococcus aureus as it competes with phagocytic cells for available IgG-Fc sites. This results in a reduction of IgG- mediated opsonization. The domains D and E of protein A are able to bind to the Fab fragments of immunoglobulins through variable (V) regions. The Fv-binding sites enable protein A to cross-link membrane IgM on B cells and therefore mediate the activation of these cells, which confers a superantigen function to protein A. Several features of the interactions of protein A with host B lymphocytes are similar to those of superantigens for T lymphocytes, which can cause various inflammatory diseases, such as toxic shock syndrome and food poisoning. Furthermore it was shown that the staphylococcal protein A is able to activate the classical complement pathway. This activation depends on the binding of a VH3+ IgM molecule to protein A, which results in the generation of an inflammatory reaction. The protein A induced complement activation is another factor, which contributes to the staphylococcal virulence. Moreover, invasive Staphylococcus aureus disease are often associated with the complication of endovascular infections. In order to cause this kind of complication staphylococci must first adhere to endovascular foci and colonize these tissues. One of the factors released by endothelial cells and by platelets is the von Willbrrand factor (vWF). This factor mediates the adhesion of platelets at damaged endothelial sites. It was shown that vWF binds to and also promotes the adhesion of staphylococcal cells to vWF-absorbed surfaces. It could also be demonstrated that the recognition of vWF is mediated by the staphylococcal protein A. Protein A defective mutants have shown reduced virulence in murine models. These observations can be explained most likely by the antiphagocytic effect of protein A binding IgG Fc fragments[8].


Medical implication

Staphylococcus aureus is known to be a dangerous pathogen, which is responsible for food poisoning and localized or generalized infections. This bacterium is often associated to multi-resistant germ to antibiotics, and it causes problems in medical field. The pathogenicity of S. aureus is due to protein A, which is one of various other important virulence factors. Protein A permits the inhibition of the phagocytosis, the process of vesicular internalization of solid particles, thanks to the interaction with mammal antibodies. In a normal case of phagocytosis, bacteria are eliminated during intracellular digestion, thanks to hydrolytic enzymes of phagocytes. At first, the particle is recognized and sticks to the phagocyte. The recognition is possible thanks to phagocyte membrane receptors, which recognize the Fc domain of immunoglobulins. Then the bacteria enter into the phagocyte by a process of endocytosis. The intracellular digestion takes place, and the bacteria are degraded by enzymes. Finally, cell fragments are removed by exocytosis. If S.aureus is located in the serum, the interaction between the Fc domain and protein A leads to the attachment in a wrong direction of the IgG to the bacteria: then, the recognition is not allowed and the cascade of reactions necessary for phagocytosis does not occur. By stopping this process and through other virulence factors, bacterial colonization is allowed through the growth and dissemination of bacteria into the organism[9].


Protein A in laboratories

Application in purification of antibodies

Thanks to its properties against immunoglobulins, protein A is used in research in technics of purification of antibodies; this kind of purification is called “Class-specific Affinity”. To accomplish the purification of IgG, the IgG-binding protein is immobilized onto a solid supports, like porous resins (for example, beaded agarose) or magnetic beads. This protein is often used, because it has an advantage: there are no orientation problems thanks to the 5 binding domains of protein A with IgG. But this protein is relatively specific to IgG; for example, it binds very poorly or not to all IgM. In order to bind other antibody targets, the use of other proteins is required, such as protein L or G, which have different binding properties. Then, the antibodies which are purified can be used to probe the specific antigen in Western blotting, ELISA (enzyme-linked immunosorbent assay) or other applications[10].


References

  1. ↑ Peterson, P. K., Verhoef, J., Sabath, L. D., & Quie, P. G. (1977). Effect of protein A on staphylococcal opsonization. Infection and immunity, 15(3), 760-4
  2. ↑ Hjelm, H., Sjödahl, J., & Sjöquist, J. (1975). Immunologically active and structurally similar fragments of protein A from Staphylococcus aureus. European journal of biochemistry / FEBS, 57(2), 395-403
  3. ↑ Bronner, S., Monteil, H., & Prévost, G. (2004). Regulation of virulence determinants in Staphylococcus aureus: complexity and applications. FEMS microbiology reviews, 28(2), 183-200
  4. ↑ PDB ID: 1SS1; S.Sato, T.L.Religa, V.Daggett, A.R. Fersht (2004) Testing protein-folding simulations by experiment: B domain of protein A. Proc.Natl.Acad.Sci.USA 101: 6952-6956
  5. ↑ Hartleib, J., Köhler, N., Dickinson, R. B., Chhatwal, G. S., Sixma, J. J., M, O., Foster, T. J., et al. (2000). Protein A is the von Willebrand factor binding protein on Staphylococcus aureus, 2149-2156
  6. ↑ Uhlén, M., Guss, B., Nilsson, B., Götz, F., & Lindberg, M. (1984). Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci. Journal of bacteriology, 159(2), 713-9
  7. ↑ O'Seaghdha, M., van Schooten, C. J., Kerrigan, S. W., Emsley, J., Silverman, G. J., Cox, D., Lenting, P. J. and Foster, T. J. (2006), Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions. FEBS Journal, 273: 4831–4841
  8. ↑ Hartleib, J., Köhler, N., Dickinson, R. B., Chhatwal, G. S., Sixma, J. J., M, O., Foster, T. J., et al. (2000). Protein A is the von Willebrand factor binding protein on Staphylococcus aureus, 2149-2156
  9. ↑ http://en.wikipedia.org/wiki/Protein_A?oldid=252478781
  10. ↑ http://www.piercenet.com/browse.cfm?fldID=4E032172-5056-8A76-4EAE-8D395D2DCDA3
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