Citrullination

PAD subtypes

PADs are found in chordates but not in lower animals. In mammals five PAD isotypes – PAD1, PAD2, PAD3, PAD4 and PAD6 – have been found. PAD5 was thought to be a unique isotype in humans, however it was shown to be homologous to PAD4. These isotypes differ in terms of their tissue and cellular distributions.

PAD1 expression has been detected in epidermis and the uterus, and it acts in citrullination of keratin and filaggrin, key components of keratinocytes.

PAD2 is expressed at a high level in the central nervous system, including the eye and brain, as well as skeletal muscle and the spleen. PAD transcripts have been found in the C57BL6/J mouse eyes as early as embryonic day 14.5. PAD2 has also been shown to interact with vimentin in skeletal muscle and macrophages, causing the filaments to disassemble, suggesting a role in apoptosis.

One of PAD2's target substrates is myelin basic protein. In the normal retina, deimination is found in nearly all the retinal layers, including the photoreceptors. Deimination has been also reported in neuronal cells, such as astrocytes, microglia and oligodendrocytes, Schwann cells and neurons. Methylation and phosphorylation of myelin basic protein are active during the process of myelinogenesis. In early development of the central nervous system of the embryo, deimination of myelin basic protein plays a major role in myelin assembly. In adults, deimination myelin basic protein occurs in demyelinating diseases such as multiple sclerosis. Myelin basic protein may affect different cell types in each case.

PAD3 expression has been linked to sheep wool modification. Citrullination of trichohyalin allows it to bind and cross-link keratin filaments, directing growth of the wool fiber.

PAD4 regulates gene expression through histone modifications. DNA is wrapped around histones, and the histone proteins can control DNA expression when chemical groups are added and removed. This process is known as post-translational processing or post-translational modification, because it takes place on the protein after the DNA is translated. The role of post-translational processing in gene regulation is the subject of the growing field of study, epigenetics. One modification mechanism is methylation. A methyl group binds to an arginine on the histone protein, altering DNA binding to the histone and allowing transcription to take place. When PAD converts arginine to citrulline on a histone, it blocks further methylation of the histone, inhibiting transcription. The main isotype for this is PAD4, which deiminates arginines and/or monomethylated arginines on histones 3 and 4, turning off the effects of arginine methylation.

Autoimmune diseases

In rheumatoid arthritis and other autoimmune diseases, such as psoriatic arthritis, systemic lupus erythematosus and Sjögren's syndrome, autoantibodies often attack citrullinated proteins. The presence of anti-citrullinated protein antibody is a standard test for rheumatoid arthritis, and it is associated with more severe disease. Citrullinated proteins are also found in the cellular debris accompanying the destruction of cells in alzheimer disease, and after smoking cigarettes. So citrullination seems to be part of the mechanism that stimulates the immune system in autoimmune disease. However, citrullinated proteins can also be found in healthy colon mucosa.

Detection of citrullinated peptides and proteins

Citrullinated peptides and proteins can be detected using antibodies targeting the citrullinated residues, or detected using mass spectrometry-based proteomics technologies. Citrullination of arginine results in a monoisotopic mass increase of +0.984016 Da, which can be measured with mass spectrometry. The mass shift is close to the mass difference between the different peptide isotopes of +1.008665 which can be mistaken for a citrullinated peptide, especially on low-resolution instruments. However, this is less of an issue with modern high resolution/high accuracy mass spectrometers. Furthermore, the mass shift is identical to the mass shift caused by deamidation of the amino acid asparagine or glutamine side chain, which are common modifications.

Citrulline residues can be chemically modified with butanedione or by biotinylation prior to analysis, leading to a different mass shift, and this strategy has successfully been used to facilitate identification by mass spectrometry.

Another approach is to utilize the neutral loss of isocyanic acid (HNCO) from citrulline residues when submitted to low energy collision induced dissociation fragmentation in mass spectrometers. The loss causes a mass shift of −43.0058 Da, which can be utilized by mass spectrometers to predominantly select citrullinated peptides for fragmentation (sequencing).

Finally, the loss of positive charge at physiological pH caused by citrullination can be utilized. Prior to bottom-up proteomics analysis, proteins are enzymatically cleaved into peptides. Commonly the protease trypsin is used, which cleaves after the positively charged arginine and lysine residues. However, trypsin is unable to cleave after a citrulline residue which is neutral. A missed cleavage after a citrulline residue together with the correct mass shift can be used as a specific and sensitive marker for citrullination, and the strategy is compatible with standard bottom-up proteomics workflows.

The first comprehensive textbook on deimination was published in 2014, while the second edition was published in 2017.

References

References

1. (March 2007). "Technical and diagnostic performance of 6 assays for the measurement of citrullinated protein/peptide antibodies in the diagnosis of rheumatoid arthritis". Clinical Chemistry.
2. (2008). "Antibodies to mutated citrullinated vimentin for diagnosing rheumatoid arthritis in anti-CCP-negative patients and for monitoring infliximab therapy". [[Arthritis Research & Therapy]].
3. (August 2007). "Clinical evaluation of anti-mutated citrullinated vimentin by ELISA in rheumatoid arthritis". The Journal of Rheumatology.
4. Vossenaar, Erik R.. (November 2003). "PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease". BioEssays.
5. Visel, Axel. (January 2004). "GenePaint.org: an atlas of gene expression patterns in the mouse embryo". Nucleic Acids Research.
6. Bhattacharya, Sanjoy. (May 2009). "Retinal deimination in aging and disease". IUBMB Life.
7. Harauz, G. (February 2007). "A tale of two citrullines—structural and functional aspects of myelin basic protein deimination in health and disease". Neurochemical Research.
8. Cuthbert, Graeme. (September 2, 2004). "Histone deimination antagonizes arginine methylation". Cell.
9. (October 2022). "Delayed puberty, gonadotropin abnormalities and subfertility in male Padi2/Padi4 double knockout mice". Reprod Biol Endocrinol.
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12. (2013). "Autoantibodies: Double Agents in Human Disease". Science Translational Medicine.
13. (2013). "Erosive Rheumatoid Arthritis is Associated with Antibodies That Activate PAD4 by Increasing Calcium Sensitivity". Science Translational Medicine.
14. (2005). "Anti-citrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis: Specificity and relation with rheumatoid factor". Autoimmunity Reviews.
15. [http://www.arthritistoday.org/about-arthritis/types-of-arthritis/rheumatoid-arthritis/who-gets-ra-and-why/causes-of-rheumatoid-arthritis/ra-and-smoking.php Can Smoking Trigger Autoimmunity in RA?] {{Webarchive. link. (2014-05-22 Scientists seek to connect the dots between smoking and rheumatoid arthritis, By Debra Dreger)
16. (2017). "Proteome Analysis of Rheumatoid Arthritis Gut Mucosa". Journal of Proteome Research.
17. (2011-06-15). "Modification of citrulline residues with 2,3-butanedione facilitates their detection by liquid chromatography/mass spectrometry". Rapid Communications in Mass Spectrometry.
18. (2013-11-01). "Specific biotinylation and sensitive enrichment of citrullinated peptides". Analytical and Bioanalytical Chemistry.
19. (2011-02-01). "On-line liquid chromatography neutral loss-triggered electron transfer dissociationmass spectrometry for the targeted analysis of citrullinated peptides". Anal. Methods.
20. (2009-04-01). "Neutral loss of isocyanic acid in peptide CID spectra: A novel diagnostic marker for mass spectrometric identification of protein citrullination". Journal of the American Society for Mass Spectrometry.
21. (2014). "Protein Deimination in Human Health and Disease". Springer.
22. (2017). "Protein Deimination in Human Health and Disease, Second Edition". Springer.