Protein A

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Protein A, Ig-binding domain
SCOP2
1DEE / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1dee​, 1l6x​

Protein A is a 42

opsonization and phagocytosis.[3]

History

As a by-product of his work on type-specific staphylococcus antigens, Verwey reported in 1940 that a protein fraction prepared from extracts of these bacteria non-specifically precipitated rabbit antisera raised against different staphylococcus types.[4] In 1958, Jensen confirmed Verwey's finding and showed that rabbit pre-immunization sera as well as normal human sera bound to the active component in the staphylococcus extract; he designated this component Antigen A (because it was found in fraction A of the extract) but thought it was a polysaccharide.[5] The misclassification of the protein was the result of faulty tests,[6] but it was not long thereafter (1962) that Löfkvist and Sjöquist corrected the error and confirmed that Antigen A was in fact a surface protein on the bacterial wall of certain strains of S. aureus.[7] The Bergen group from Norway named the protein "Protein A" after the antigen fraction isolated by Jensen.[8]

Protein A antibody binding

It has been shown via crystallographic refinement that the primary binding site for protein A is on the Fc region, between the CH2 and CH3 domains.[9] In addition, protein A has been shown to bind human IgG molecules containing IgG F(ab')2 fragments from the human VH3 gene family.[10]

Protein A can bind with strong affinity to the Fc portion of immunoglobulin of certain species as shown in the below table.[11]

Species Subclass Binding
Human IgA variable
IgD weak or none
IgE weak or none
IgG1 strong
IgG2 strong
IgG3 weak or none
IgG4 strong
IgM variable
Avian egg yolk IgY weak or none
Bovine medium
Canine medium
Goat weak or none
Guinea pig IgG1 strong
Hamster weak
Horse medium
Koala weak or none
Llama weak or none
Monkey (rhesus) strong
Murine IgG1 weak
IgG2a strong
IgG2 medium to strong
IgG3 medium
IgM variable
Pig medium to strong
Rabbit strong
Rat IgG1 weak or none
IgG2a weak or none
IgG2b weak or none
IgG3 weak
Sheep weak or none

Other antibody binding proteins

In addition to protein A, other immunoglobulin-binding bacterial proteins such as protein G, protein A/G and protein L are all commonly used to purify, immobilize or detect immunoglobulins.

Role in pathogenesis

As a pathogen, Staphylococcus aureus utilizes protein A, along with a host of other proteins and surface factors, to aid its survival and virulence. To this end, protein A plays a multifaceted role:

  1. By binding the Fc portion of antibodies, protein A renders them inaccessible to the opsonins, thus impairing phagocytosis of the bacteria via immune cell attack.
  2. Protein A facilitates the adherence of S. aureus to human von Willebrand factor (vWF)-coated surfaces, thus increasing the bacteria's infectiousness at the site of skin penetration.
  3. Protein A can inflame lung tissue by binding to
    tumor necrosis factor 1
    (TNFR-1) receptors. This interaction has been shown to play a key role in the pathogenesis of staphylococcal pneumonia.
  4. Protein A has been shown to cripple humoral (antibody-mediated) immunity which in turn means that individuals can be repeatedly infected with S. aureus since they cannot mount a strong antibody response.
  5. Protein A has been shown to promote the formation of biofilms both when the protein is covalently linked to the bacterial cell wall as well as in solution.[12]

Protein A helps inhibit phagocytic engulfment and acts as an immunological disguise. Higher levels of protein A in different strains of S. aureus have been associated with nasal carriage of this bacteria.[13]

Mutants of S. aureus lacking protein A are more efficiently phagocytosed in vitro, and mutants in infection models have diminished virulence.[14]

Production

Protein A is produced and purified in industrial fermentation for use in immunology, biological research and industrial applications (see below). Natural (or native) protein A can be cultured in Staphylococcus aureus and contains the five homologous antibody binding regions described above and a C-terminal region for cell wall attachment. Today, protein A is more commonly produced recombinantly in Escherichia coli. (Brevibacillus has also been shown to be an effective host.[15]) Recombinant versions of protein A also contain the five homologous antibody binding domains but may vary in other parts of the structure in order to facilitate coupling to porous substrates.[16] Engineered versions of the protein are also available, the first of which was rProtein A, B4, C-CYS.[17] Engineered versions are multimers (typically tetramers, pentamers or hexamers) of a single domain which has been modified to improve usability in industrial applications.

Research

Protein A is often coupled to other molecules such as a

fluorescent dye, enzymes, biotin, colloidal gold or radioactive iodine without affecting the antibody binding site. Examples including protein A–gold (PAG) stain is used in immunogold labelling, fluorophore coupled protein A for immunofluorescence, and DNA docking strand coupled protein A for DNA-PAINT imaging.[18] It is also widely utilized coupled to magnetic, latex and agarose
beads.

Protein A is often immobilized onto a solid support and used as reliable method for purifying total IgG from crude protein mixtures such as

serum or ascites fluid, or coupled with one of the above markers to detect the presence of antibodies. The first example of protein A being coupled to a porous bead for purification of IgG was published in 1972.[19] Immunoprecipitation
studies with protein A conjugated to beads are also commonly used to purify proteins or protein complexes indirectly through antibodies against the protein or protein complex of interest.

Role in industrial purification of antibodies

This process flow diagram shows how monoclonal antibodies are typically purified at industrial scale.

The first reference in the literature to a commercially available protein A chromatography resin appeared in 1976.

monoclonal antibodies (mAbs) from harvest cell culture supernatant.[21] The choice of protein A as the preferred method is due to the high purity and yield which are easily and reliably achieved. This forms the basis for a general antibody purification "platform" which simplifies manufacturing operations and reduces the time and effort required to develop purification processes.[22] A typical mAb purification process is shown at right. Albeit the long history of protein A chromatography for the production of antibodies, the process is still being improved today. Continuous chromatography, more precisely periodic counter-current chromatography
, enormously increases the productivity of the purification step.

References

  1. PMID 10805799
    .
  2. PMID 10754313
    .
  3. PMID 28031339
    .
  4. PMID 19870987
    .
  5. PMID 17504410
    .
  6. ISBN 9780120224333
    .
  7. doi:10.1111/j.1699-0463.1962.tb04908.x
    .
  8. PMID 14185494
    .
  9. PMID 7236608
    .
  10. S2CID 35812099
    .
  11. ^ Affinity Chromatography (PDF). Vol. 1: Antibodies (AF ed.). GE Healthcare. 2016. p. 48.
  12. ^ "A Pathogen's Swiss Army Knife". Small Things Considered. Retrieved 2016-08-25.
  13. PMID 21338050
    .
  14. PMID 12719481
    .
  15. ^ Kosugi A, JP, Yajima K, JP (November 18, 2014), United States Patent: 8889389 - Process for producing protein A-like protein with use of Brevibacillus genus bacterium, retrieved 2016-08-26
  16. ^ "usp31nf26s1_c130". www.uspbpep.com. General Chapters: PROTEIN A QUALITY ATTRIBUTES. Retrieved 2016-08-26.
  17. ^ Hober S (June 12, 2012), United States Patent: 8198404 - Mutated immunoglobulin-binding protein, retrieved 2016-08-26
  18. S2CID 58547594
    .
  19. S2CID 8733702
    .
  20. PMID 12109
    .
  21. PMID 17046339
    .
  22. PMID 20647768
    .
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