Citrullination

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The chemical conversion of arginine to citrulline, known as citrullination or deimination. Citrullination.svg
The chemical conversion of arginine to citrulline, known as citrullination or deimination.

Citrullination or deimination is the conversion of the amino acid arginine in a protein into the amino acid citrulline. Citrulline is not one of the 20 standard amino acids encoded by DNA in the genetic code. Instead, it is the result of a post-translational modification. Citrullination is distinct from the formation of the free amino acid citrulline as part of the urea cycle or as a byproduct of enzymes of the nitric oxide synthase family.

Contents

Enzymes called arginine deiminases (ADIs) catalyze the deimination of free arginine, while protein arginine deiminases or peptidylarginine deiminases (PADs) replace the primary ketimine group (>C=NH) by a ketone group (>C=O). Arginine is positively charged at a neutral pH, whereas citrulline has no net charge. This increases the hydrophobicity of the protein, which can lead to changes in protein folding, affecting the structure and function.

The immune system can attack citrullinated proteins, leading to autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). Fibrin and fibrinogen may be favored sites for arginine deimination within rheumatoid joints. Test for presence of anti-citrullinated protein (ACP) antibodies are highly specific (88–96%) for rheumatoid arthritis, about as sensitive as rheumatoid factor (70–78%) for diagnosis of RA, and are detectable from even before the onset of clinical disease. [1]

Citrullinated vimentin may be an autoantigen in RA and other autoimmune diseases, and is used to study RA. Moreover, antibodies against mutated citrullinated vimentin (MCV) may be useful for monitoring effects of RA therapy. [2] An ELISA system utilises genetically modified citrullinated vimentin (MCV), a naturally occurring isoform of vimentin to improve the performance of the test. [3]

In the reaction from arginine to citrulline, one of the terminal nitrogen atoms of the arginine side chain is replaced by an oxygen. Thus, arginine's positive charge (at physiological pH) is removed, altering the protein's tertiary structure. The reaction uses one water molecule and yields ammonia as a side-product:

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. [4] PAD5 was thought to be a unique isotype in humans, however it was shown to be homologous to PAD4. [4] 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. [4]

PAD2 is expressed at a high level in the central nervous system (CNS), 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. [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. [4]

One of PAD2's target substrates is myelin basic protein (MBP). 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. [6] Methylation and phosphorylation of MBP are active during the process of myelinogenesis. In early CNS development of the embryo, MBP deimination plays a major role in myelin assembly. In adults, MBP deimination is found in demyelination diseases such as multiple sclerosis. MBP may affect different cell types in each case. [7]

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. [4]

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 (CH3) 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. [8] [9] 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. [10]

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. [11] [12] [13] [14] [15] [16]

The first comprehensive textbook on deimination was published in 2014. [17]

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. [18] [19]

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). [20] [21]

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.

Related Research Articles

<span class="mw-page-title-main">Arginine</span> Amino acid

Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the amino and guanidino groups are protonated, resulting in a cation. Only the l-arginine (symbol Arg or R) enantiomer is found naturally. Arg residues are common components of proteins. It is encoded by the codons CGU, CGC, CGA, CGG, AGA, and AGG. The guanidine group in arginine is the precursor for the biosynthesis of nitric oxide. Like all amino acids, it is a white, water-soluble solid.

<span class="mw-page-title-main">Citrulline</span> Chemical compound

The organic compound citrulline is an α-amino acid. Its name is derived from citrullus, the Latin word for watermelon. Although named and described by gastroenterologists since the late 19th century, it was first isolated from watermelon in 1914 by Japanese researchers Yotaro Koga and Ryo Odake and further codified by Mitsunori Wada of Tokyo Imperial University in 1930. It has the formula H2NC(O)NH(CH2)3CH(NH2)CO2H. It is a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia by converting it into urea. Citrulline is also produced as a byproduct of the enzymatic production of nitric oxide from the amino acid arginine, catalyzed by nitric oxide synthase.

<span class="mw-page-title-main">Post-translational modification</span> Biological processes

Post-translational modification (PTM) is the covalent process of changing proteins following protein biosynthesis. PTMs may involve enzymes or occur spontaneously. Proteins are created by ribosomes translating mRNA into polypeptide chains, which may then change to form the mature protein product. PTMs are important components in cell signalling, as for example when prohormones are converted to hormones.

Rheumatoid factor (RF) is the autoantibody that was first found in rheumatoid arthritis. It is defined as an antibody against the Fc portion of IgG and different RFs can recognize different parts of the IgG-Fc. RF and IgG join to form immune complexes that contribute to the disease process such as chronic inflammation and join destruction at the synovium and cartilage.

<span class="mw-page-title-main">Myelin basic protein</span>

Myelin basic protein (MBP) is a protein believed to be important in the process of myelination of nerves in the nervous system. The myelin sheath is a multi-layered membrane, unique to the nervous system, that functions as an insulator to greatly increase the velocity of axonal impulse conduction. MBP maintains the correct structure of myelin, interacting with the lipids in the myelin membrane.

<span class="mw-page-title-main">Histone H4</span> One of the five main histone proteins involved in the structure of chromatin

Histone H4 is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H4 is involved with the structure of the nucleosome of the 'beads on a string' organization. Histone proteins are highly post-translationally modified. Covalently bonded modifications include acetylation and methylation of the N-terminal tails. These modifications may alter expression of genes located on DNA associated with its parent histone octamer. Histone H4 is an important protein in the structure and function of chromatin, where its sequence variants and variable modification states are thought to play a role in the dynamic and long term regulation of genes.

<span class="mw-page-title-main">Deamidation</span>

Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino acids asparagine or glutamine is removed or converted to another functional group. Typically, asparagine is converted to aspartic acid or isoaspartic acid. Glutamine is converted to glutamic acid or pyroglutamic acid (5-oxoproline). In a protein or peptide, these reactions are important because they may alter its structure, stability or function and may lead to protein degradation. The net chemical change is the addition of a water group and removal of an ammonia group, which corresponds to a +1 (0.98402) Da mass increase. Although deamidation occurs on glutamine, glycosylated asparagine and other amides, these are negligible under typical proteolysis conditions.

<span class="mw-page-title-main">Histone-modifying enzymes</span> Type of enzymes

Histone-modifying enzymes are enzymes involved in the modification of histone substrates after protein translation and affect cellular processes including gene expression. To safely store the eukaryotic genome, DNA is wrapped around four core histone proteins, which then join to form nucleosomes. These nucleosomes further fold together into highly condensed chromatin, which renders the organism's genetic material far less accessible to the factors required for gene transcription, DNA replication, recombination and repair. Subsequently, eukaryotic organisms have developed intricate mechanisms to overcome this repressive barrier imposed by the chromatin through histone modification, a type of post-translational modification which typically involves covalently attaching certain groups to histone residues. Once added to the histone, these groups elicit either a loose and open histone conformation, euchromatin, or a tight and closed histone conformation, heterochromatin. Euchromatin marks active transcription and gene expression, as the light packing of histones in this way allows entry for proteins involved in the transcription process. As such, the tightly packed heterochromatin marks the absence of current gene expression.

<span class="mw-page-title-main">Protein-arginine deiminase</span>

In enzymology, a protein-arginine deiminase (EC 3.5.3.15) is an enzyme that catalyzes a form of post translational modification called arginine de-imination or citrullination:

<span class="mw-page-title-main">PADI4</span> Protein-coding gene in the species Homo sapiens

Protein-arginine deiminase type-4, is a human protein which in humans is encoded by the PADI4 gene. The protein as an enzyme, specifically protein-arginine deiminase, a type of hydrolase.

<span class="mw-page-title-main">Protein phosphorylation</span> Process of introducing a phosphate group on to a protein

Protein phosphorylation is a reversible post-translational modification of proteins in which an amino acid residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Phosphorylation alters the structural conformation of a protein, causing it to become either activated or deactivated, or otherwise modifying its function. Approximately 13000 human proteins have sites that are phosphorylated.

<span class="mw-page-title-main">Anti–citrullinated protein antibody</span> Autoantibodies

Anti-citrullinated protein antibodies (ACPAs) are autoantibodies that are directed against peptides and proteins that are citrullinated. They are present in the majority of patients with rheumatoid arthritis. Clinically, cyclic citrullinated peptides (CCP) are frequently used to detect these antibodies in patient serum or plasma.

Detection of autoantibodies against mutated citrullinated vimentin is part of rheumatoid arthritis (RA) diagnostics, especially in sera negative for rheumatoid factor. Anti-MCV antibodies are a member of the ACPA family, a group of the so-called antibodies to citrullinated protein/peptide antigens.

The New South Wales Systems Biology Initiative, directed by Marc Wilkins is a non-profit facility within the School of Biotechnology and Biomolecular Sciences at the University of New South Wales. Their focus is undertaking basic and applied research in the development and application of bioinformatics for genomics and proteomics.

Rheumatoid lung disease is a disease of the lung associated with RA, rheumatoid arthritis. Rheumatoid lung disease is characterized by pleural effusion, pulmonary fibrosis, lung nodules and pulmonary hypertension. Common symptoms associated with the disease include shortness of breath, cough, chest pain and fever. It is estimated that about one quarter of people with rheumatoid arthritis develop this disease, which are more likely to develop among elderly men with a history of smoking.

<span class="mw-page-title-main">Homocitrulline</span> Chemical compound

L-Homocitrulline is an amino acid and a metabolite of ornithine in mammalian metabolism. The amino acid can be detected in larger amounts in the urine of individuals with urea cycle disorders. At present, it is thought that the depletion of the ornithine supply causes the accumulation of carbamyl-phosphate in the urea cycle which may be responsible for the enhanced synthesis of homocitrulline and homoarginine. Both amino acids can be detected in urine. Amino acid analysis allows for the quantitative analysis of these amino acid metabolites in biological fluids such as urine or blood.

Protein methylation is a type of post-translational modification featuring the addition of methyl groups to proteins. It can occur on the nitrogen-containing side-chains of arginine and lysine, but also at the amino- and carboxy-termini of a number of different proteins. In biology, methyltransferases catalyze the methylation process, activated primarily by S-adenosylmethionine. Protein methylation has been most studied in histones, where the transfer of methyl groups from S-adenosyl methionine is catalyzed by histone methyltransferases. Histones that are methylated on certain residues can act epigenetically to repress or activate gene expression.

<span class="mw-page-title-main">Lars Klareskog</span>

Lars Klareskog is a Swedish physician, immunologist, and rheumatologist, known for research into the genetics of autoimmune diseases such as rheumatoid arthritis (RA).

H3R8me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 8th arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

Epigenetics of autoimmune disorders is the role that epigenetics play in autoimmune diseases. Autoimmune disorders are a diverse class of diseases that share a common origin. These diseases originate when the immune system becomes dysregulated and mistakenly attacks healthy tissue rather than foreign invaders. These diseases are classified as either local or systemic based upon whether they affect a single body system or if they cause systemic damage.

References

  1. ↑ Coenen D, Verschueren P, Westhovens R, Bossuyt X (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. 53 (3): 498–504. doi: 10.1373/clinchem.2006.078063 . PMID   17259232.
  2. ↑ Nicaise Roland P, Grootenboer Mignot S, Bruns A, et al. (2008). "Antibodies to mutated citrullinated vimentin for diagnosing rheumatoid arthritis in anti-CCP-negative patients and for monitoring infliximab therapy". Arthritis Research & Therapy . 10 (6): R142. doi:10.1186/ar2570. PMC   2656247 . PMID   19077182.
  3. ↑ Soós L, Szekanecz Z, Szabó Z, et al. (August 2007). "Clinical evaluation of anti-mutated citrullinated vimentin by ELISA in rheumatoid arthritis". The Journal of Rheumatology. 34 (8): 1658–63. PMID   17611988. Archived from the original on 2008-02-20. Retrieved 2009-10-07.
  4. 1 2 3 4 5 Vossenaar, Erik R.; Albert J.W. Zendman; Walther J. van Venrooij; Ger J.M. Pruijn (November 2003). "PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease". BioEssays. 25 (11): 1106–1118. doi:10.1002/bies.10357. PMID   14579251.
  5. ↑ Visel, Axel; Thaller, Eichele (January 2004). "GenePaint.org: an atlas of gene expression patterns in the mouse embryo". Nucleic Acids Research. 32 (Database issue): D552–6. doi:10.1093/nar/gkh029. PMC   308763 . PMID   14681479.
  6. ↑ Bhattacharya, Sanjoy (May 2009). "Retinal deimination in aging and disease". IUBMB Life. 61 (5): 504–509. doi: 10.1002/iub.184 . PMID   19391158.
  7. ↑ Harauz, G; Mussee (February 2007). "A tale of two citrullines—structural and functional aspects of myelin basic protein deimination in health and disease". Neurochemical Research. 32 (2): 137–158. doi:10.1007/s11064-006-9108-9. PMID   16900293.
  8. ↑ Cuthbert, Graeme; Daujat, Snowden; Erdjument-Bromage, Hagiwara; Yamada, Schneider; Gregory, Tempst; Bannister, Kouzarides (September 2, 2004). "Histone deimination antagonizes arginine methylation". Cell. 118 (5): 545–553. doi: 10.1016/j.cell.2004.08.020 . PMID   15339660.
  9. ↑ Sams, K.L; Mukai, C; Marks, B.A; Mittal, C; Demeter, E.A; Nelissen, S; Grenier, J.K; Tate, A.E; Ahmed, F; Coonrod, S.A (October 2022). "Delayed puberty, gonadotropin abnormalities and subfertility in male Padi2/Padi4 double knockout mice". Reprod Biol Endocrinol. 20 (1): 150. doi:10.1186/s12958-022-01018-w. PMC   9555066 . PMID   36224627.
  10. ↑ Kouzarides, T (November 2007). "SnapShot: Histone-modifying enzymes". Cell. 131 (4): 822–822.e1. doi: 10.1016/j.cell.2007.11.005 . PMID   18022374.
  11. ↑ Enzyme-activating antibodies revealed as marker for most severe form of rheumatoid arthritis, Science Daily, May 22, 2013
  12. ↑ Acharya, N. K.; Nagele, E. P.; Han, M.; Nagele, R. G. (2013). "Autoantibodies: Double Agents in Human Disease". Science Translational Medicine. 5 (186): 186fs19. doi:10.1126/scitranslmed.3006288. PMID   23698377.
  13. ↑ Darrah, E.; Giles, J. T.; Ols, M. L.; Bull, H. G.; Andrade, F.; Rosen, A. (2013). "Erosive Rheumatoid Arthritis is Associated with Antibodies That Activate PAD4 by Increasing Calcium Sensitivity". Science Translational Medicine. 5 (186): 186ra65. doi:10.1126/scitranslmed.3005370. PMC   3740946 . PMID   23698378.
  14. ↑ Vander Cruyssen, B.; Peene, I.; Cantaert, T.; Hoffman, I.E.A.; De Rycke, L.; Veys, E.M.; De Keyser, F. (2005). "Anti-citrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis: Specificity and relation with rheumatoid factor". Autoimmunity Reviews. 4 (7): 468–474. doi:10.1016/j.autrev.2005.04.018. PMID   16137613.
  15. ↑ Can Smoking Trigger Autoimmunity in RA? Archived 2014-05-22 at the Wayback Machine Scientists seek to connect the dots between smoking and rheumatoid arthritis, By Debra Dreger
  16. ↑ Bennike, Tue Bjerg; Ellingsen, Torkell; Glerup, Henning; Bonderup, Ole Kristian; Carlsen, Thomas Gelsing; Meyer, Michael Kruse; Bøgsted, Martin; Christiansen, Gunna; Birkelund, Svend; Andersen, Vibeke; Stensballe, Allan (2017). "Proteome Analysis of Rheumatoid Arthritis Gut Mucosa". Journal of Proteome Research. 16 (1): 346–354. doi:10.1021/acs.jproteome.6b00598. PMID   27627584.
  17. ↑ Nicholas, AP; Bhattacharya, SK (2014). Protein Deimination in Human Health and Disease. New York: Springer. ISBN   978-1-4614-8317-5.
  18. ↑ De Ceuleneer, Marlies; De Wit, Vanessa; Van Steendam, Katleen; Van Nieuwerburgh, Filip; Tilleman, Kelly; Deforce, Dieter (2011-06-15). "Modification of citrulline residues with 2,3-butanedione facilitates their detection by liquid chromatography/mass spectrometry". Rapid Communications in Mass Spectrometry. 25 (11): 1536–1542. doi:10.1002/rcm.5015. ISSN   1097-0231. PMID   21594927.
  19. ↑ Tutturen, Astrid E. V.; Holm, Anders; Fleckenstein, Burkhard (2013-11-01). "Specific biotinylation and sensitive enrichment of citrullinated peptides". Analytical and Bioanalytical Chemistry. 405 (29): 9321–9331. doi:10.1007/s00216-013-7376-1. ISSN   1618-2642. PMID   24081567.
  20. ↑ Creese, Andrew J.; Grant, Melissa M.; Chapple, Iain L. C.; Cooper, Helen J. (2011-02-01). "On-line liquid chromatography neutral loss-triggered electron transfer dissociationmass spectrometry for the targeted analysis of citrullinated peptides". Anal. Methods. 3 (2): 259–266. doi: 10.1039/c0ay00414f . ISSN   1759-9679. PMID   32938022.
  21. ↑ Hao, Gang; Wang, Danchen; Gu, Jane; Shen, Qiuying; Gross, Steven S.; Wang, Yanming (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. 20 (4): 723–727. doi:10.1016/j.jasms.2008.12.012. ISSN   1044-0305. PMC   2786913 . PMID   19200748.
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