Citrullination
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Citrullination or deimination is the conversion of the
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
arginine 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 a ...
in a protein into the amino acid
citrulline 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 1 ...
. Citrulline is not one of the 20 standard amino acids encoded by DNA in the
genetic code The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
. Instead, it is the result of a
post-translational modification Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribos ...
. Citrullination is distinct from the formation of the free amino acid citrulline as part of the
urea cycle The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions that produces urea (NH2)2CO from ammonia (NH3). Animals that use this cycle, mainly amphibians and mammals, are called ureotelic. The urea cycle converts hi ...
or as a byproduct of enzymes of the
nitric oxide synthase Nitric oxide synthases () (NOSs) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule. It helps modulate vascular tone, insulin secretion, airway tone, and per ...
family. Enzymes called arginine deiminases (ADIs) catalyze the deimination of free arginine, while protein arginine deiminases or peptidylarginine deiminases (PADs) replace the primary
ketimine In organic chemistry, an imine ( or ) is a functional group or organic compound containing a carbon–nitrogen double bond (). The nitrogen atom can be attached to a hydrogen or an organic group (R). The carbon atom has two additional single bon ...
group (>C=NH) by a
ketone In organic chemistry, a ketone is a functional group with the structure R–C(=O)–R', where R and R' can be a variety of carbon-containing substituents. Ketones contain a carbonyl group –C(=O)– (which contains a carbon-oxygen double bon ...
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 Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects synovial joint, joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and ...
(RA) and multiple sclerosis (MS).
Fibrin Fibrin (also called Factor Ia) is a fibrous, non-globular protein involved in the clotting of blood. It is formed by the action of the protease thrombin on fibrinogen, which causes it to polymerize. The polymerized fibrin, together with pla ...
and
fibrinogen Fibrinogen (factor I) is a glycoprotein complex, produced in the liver, that circulates in the blood of all vertebrates. During tissue and vascular injury, it is converted enzymatically by thrombin to fibrin and then to a fibrin-based blood ...
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 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 co ...
(70–78%) for diagnosis of RA, and are detectable from even before the onset of clinical disease. 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. An
ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
system utilises genetically modified citrullinated vimentin (MCV), a naturally occurring isoform of vimentin to improve the performance of the test. In the reaction from arginine to citrulline, one of the terminal
nitrogen Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at seve ...
atoms of the arginine
side chain In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called the "main chain" or backbone. The side chain is a hydrocarbon branching element of a molecule that is attached to a ...
is replaced by an
oxygen Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements as we ...
. Thus, arginine's positive charge (at physiological pH) is removed, altering the protein's tertiary structure. The reaction uses one
water Water (chemical formula ) is an inorganic, transparent, tasteless, odorless, and nearly colorless chemical substance, which is the main constituent of Earth's hydrosphere and the fluids of all known living organisms (in which it acts as ...
molecule and yields
ammonia Ammonia is an inorganic compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogeno ...
as a side-product:


PAD subtypes

PADs are found in chordates but not in lower animals. In mammals five PAD isotypesPAD1, 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 Keratin () is one of a family of structural fibrous proteins also known as ''scleroproteins''. Alpha-keratin (α-keratin) is a type of keratin found in vertebrates. It is the key structural material making up scales, hair, nails, feathers, ...
and
filaggrin Filaggrin (filament aggregating protein) is a filament-associated protein that binds to keratin fibers in epithelial cells. Ten to twelve filaggrin units are post-translationally hydrolyzed from a large profilaggrin precursor protein during termin ...
, key components of
keratinocytes Keratinocytes are the primary type of cell found in the epidermis, the outermost layer of the skin. In humans, they constitute 90% of epidermal skin cells. Basal cells in the basal layer (''stratum basale'') of the skin are sometimes referre ...
. PAD2 is expressed at a high level in the
central nervous system The central nervous system (CNS) is the part of the nervous system consisting primarily of the brain and spinal cord. The CNS is so named because the brain integrates the received information and coordinates and influences the activity of all p ...
(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. PAD2 has also been shown to interact with
vimentin Vimentin is a structural protein that in humans is encoded by the ''VIM'' gene. Its name comes from the Latin ''vimentum'' which refers to an array of flexible rods. Vimentin is a type III intermediate filament (IF) protein that is expresse ...
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 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 ...
(MBP). In the normal
retina The retina (from la, rete "net") is the innermost, light-sensitive layer of tissue of the eye of most vertebrates and some molluscs. The optics of the eye create a focused two-dimensional image of the visual world on the retina, which then ...
, deimination is found in nearly all the retinal layers, including the photoreceptors. Deimination has been also reported in neuronal cells, such as
astrocytes Astrocytes (from Ancient Greek , , "star" + , , "cavity", "cell"), also known collectively as astroglia, are characteristic star-shaped glial cells in the brain and spinal cord. They perform many functions, including biochemical control of endo ...
,
microglia Microglia are a type of neuroglia (glial cell) located throughout the brain and spinal cord. Microglia account for about 7% of cells found within the brain. As the resident macrophage cells, they act as the first and main form of active immune ...
and
oligodendrocytes Oligodendrocytes (), or oligodendroglia, are a type of neuroglia whose main functions are to provide support and insulation to axons in the central nervous system of jawed vertebrates, equivalent to the function performed by Schwann cells in the ...
,
Schwann cells Schwann cells or neurolemmocytes (named after German physiologist Theodor Schwann) are the principal glia of the peripheral nervous system (PNS). Glial cells function to support neurons and in the PNS, also include satellite cells, olfactory en ...
and
neurons A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. ...
. Methylation and
phosphorylation In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, ...
of MBP are active during the process of
myelinogenesis Myelinogenesis is the formation and development of myelin sheaths in the nervous system, typically initiated in late prenatal neurodevelopment and continuing throughout postnatal development. Myelinogenesis continues throughout the lifespan to s ...
. 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. PAD3 expression has been linked to sheep wool modification. Citrullination of
trichohyalin Trichohyalin is a protein that in mammals is encoded by the ''TCHH'' gene. Discovery In 1903 the name ''trichohyalin'' was assigned to the granules of the inner root sheath (IRS) of hair follicles discovered by Hans Vörner. In 1986 the name was ...
allows it to bind and cross-link keratin filaments, directing growth of the wool fiber. PAD4 regulates gene expression through
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn ar ...
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 In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are " ...
. One modification mechanism is
methylation In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...
. 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. 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 Psoriatic arthritis is a long-term inflammatory arthritis that occurs in people affected by the autoimmune disease psoriasis. The classic feature of psoriatic arthritis is swelling of entire fingers and toes with a sausage-like appearance. ...
,
systemic lupus erythematosus Lupus, technically known as systemic lupus erythematosus (SLE), is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissue in many parts of the body. Symptoms vary among people and may be mild to severe. Comm ...
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 Alzheimer's disease (AD) is a neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As t ...
, 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 A mucous membrane or mucosa is a membrane that lines various cavities in the body of an organism and covers the surface of internal organs. It consists of one or more layers of epithelial cells overlying a layer of loose connective tissue. It is ...
. The first comprehensive textbook on deimination was published in 2014.


Detection of citrullinated peptides and proteins

Citrullinated peptides and proteins can be detected using antibodies targeting the citrullinated residues, or detected using
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...
-based
proteomics Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
technologies. Citrullination of arginine results in a monoisotopic mass increase of +0.984016 Da, which can be measured with
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...
. 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 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 isoaspa ...
of the
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
asparagine Asparagine (symbol Asn or N) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depro ...
or
glutamine Glutamine (symbol Gln or Q) is an α-amino acid that is used in the biosynthesis of proteins. Its side chain is similar to that of glutamic acid, except the carboxylic acid group is replaced by an amide. It is classified as a charge-neutral ...
side chain, which are common modifications. Citrulline residues can be chemically modified with
butanedione Diacetyl ( IUPAC systematic name: butanedione or butane-2,3-dione) is an organic compound with the chemical formula (CH3CO)2. It is a yellow liquid with an intensely buttery flavor. It is a vicinal diketone (two C=O groups, side-by-side). Dia ...
or by
biotinylation In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of bi ...
prior to analysis, leading to a different mass shift, and this strategy has successfully been used to facilitate identification by
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...
. 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 Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. The major alternative work ...
analysis, proteins are enzymatically cleaved into peptides. Commonly the
protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the form ...
trypsin Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the d ...
is used, which cleaves after the positively charged
arginine 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 a ...
and
lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated &minu ...
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 Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. The major alternative work ...
workflows.


References

{{Protein primary structure Post-translational modification Protein structure