Posttranslational modifications (PTMs) bring about changes in proteins and enable regulation of many cellular processes. Proteins may undergo several PTMs, such as: oxidation of amino acid side chains (prolyl, arginyl and lysyl residues); deamidation of asparaginyl and glutaminyl residues; racemization and isomerization of aspartyl, asparaginyl and prolyl residues and oxidation of cysteine sulfhydryl groups1.
Deimination refers to the conversion of protein bound arginine into citrulline (Fig. 1). It is a PTM, performed by peptidyl arginine deiminases (PADs)2. Usually, the enzymes responsible for PTMs occur in pairs catalyzing opposite reactions, for example, kinases and phosphatases for phosphorylation, acetylases and deacetylases for acetylation. However, no known enzyme exists to reverse deimination rendering the latter a long term PTM. Only a few proteins: Keratin, Myelin basic protein (MBP), Glial fibrillary acidic protein (GFAP), Vimentin, Trichohyalin, Histones (H2A, H3 and H4), Filaggrin and Fibrinogen have been reported to undergo deimination3. Recently, immunoprecipitation and mass spectrometry have identified about 36 proteins (Table 1) that potentially undergo deimination4. In the human retina, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, MBP and Myelin-associated glycoprotein (MAG) have been identified to undergo deimination4 and further independent investigation has confirmed their deimination in the brain as well5. Quantitative mass spectrometry has also identified a few additional proteins that undergo deimination6. A mass spectrometric method for detection of chemically modified citrulline residues7 suffers from limitation of completion of the chemical reaction steps with increase in peptide complexity. Chemical reaction to modify citrulline is relatively incomplete with bigger polypeptide chain proteins than with smaller peptides(For example, if MBP isoform 18.5 or a 32-amino acid synthetic peptide with a few citrullines is subjected to chemical modification (monooxime formation using 2, 3 butanedione and antipyrine) to chemically convert the citrulline into an adduct that is recognized by the modified anti-citrulline antibody, with equivalent molar ratio of protein/peptide and 2, 3 butanedione, whereas only about 10–20% of citrullines within MBP is modified or adducted, the adduct formation for the 32-mer synthetic citrulline containing peptide is about 90–100%). Although the possibility that deimination contributes to epigenetic control of gene expression8, 9 and apoptosis10 has been described, the deimination or its modulation in normal cells and tissues have not been ascribed to a specific physiological condition as yet.
Table 1. Potential citrullinated proteins in the retina/posterior ocular tissue
Swiss-Protein database accession numbers of proteins identified from anti-citruline IP product from posterior ocular tissue/retina by LC MS/MS are shown which is available at (http://us.expasy.org/sport/).
14-3-3 protein epsilon
14-3-3 protein gamma
14-3-3 protein zeta/delta
78 kDa glucose-regulated protein
Dihydropyrimidinase related protein-1
Dihydropyrimidinase related protein-2
Dihydropyrimidinase related protein-3
Glial fibriliary acidic protein, astroctye
Myelin basic protein
Myelin P0 protein
Neural cell adhesion molecule (N-CAM 140)
Neurofilament triplet H protein
Transitional endosplasmic reticulam ATPase
Tublin beta-4q chain
Voltage-dependent anion-selective channel proteir
ENZYMATIC ACITIVITY AND SPECIFICITY
Five protein deiminases, PAD1-4 and 6 have been known to exist in mammals2. Homologues of deiminases are absent in non-vertebrate organisms such as fruit flies (Drosophila melanogaster). Protein deimination in skin, muscle and epidermal tissue is catalyzed by PAD1 and PAD39, 11. PAD2 is the major deiminase in the neuronal tissues, including the eye and brain. PAD4 is nuclear and expressed ubiquitously2, 4, 12. PAD4 activation has been suggested to result in transcriptional repression13. PAD4 has been implicated in the reversal of another relatively long term PTM, protein methylation by demethylimination13, 14. Because the methylated arginines have been shown to be very poor substrates for deiminases15, including PAD4, demethylimination by PAD4 (or any other deiminase)16 remains to be conclusively established. PAD6 has been found to lack deiminase enzymatic activity15 and has been observed to be essential for oocyte cytoskeletal sheet formation and female fertility17.
Protein-bound but not free arginines are substrates of PADs2. PAD catalyzed deimination requires calcium for activation and generates ammonia as a byproduct. Conversely, Protein-bound arginines are not substrates for nitric oxide synthase (NOS), which catalyze the conversion of free arginines to free citrullines to generate nitric oxide (NO) as a by-product. The term citrullination is usually interchangeably used for protein deimination. Citrullination refers to the conversion of both free and protein bound arginines into citrulline. However, in this review we will use deimination for conversion of protein bound arginine into citrulline.
The enzymatic activity of PADs is usually measured using benzoylarginine18 employing spectrophotometry. The converted benzoylcitrulline by deiminases has been measured with or without generation of chemical derivatives19 usually employing spectrophotometry at the end to detect the appearance of a product with characteristic absorbance18, 20. In a widely used chemical method, protein bound arginine in a strong acidic environment is made to react with 2,3-butandione and antipyrine leading to the generation of an adduct in a two step reaction process21. This process of chemical modification by butanedione and antipyrine in an acidic atmosphere is very inefficient for free citrullines, and therefore it has specificity for protein bound citrullines. A specific antibody is available to detect the citrulline adduct of 2,3-butanedione and antipyrine21. Another antibody that detects protein bound unmodified citrulline22 is also available.
RETINAL DEIMINATION IN AGING AND DISEASES
In normal retina, deimination is found in nearly all the retinal layers, including the photoreceptors, as evidenced by the significant reaction of the anti-citrulline antibody (Fig. 2). Deimination has been also reported in extra-retinal neuronal cells, such as astrocytes22–28, microglia and oligodendrocytes29, 30, Schwann cells31 and neurons6, 12, 31. Astrocytes, microglia, and neurons are found in the retina whereas oligodendrocytes and Schwann cells occur in brain spinal cord and the optic nerve but not in the retina. Conditions of stress and disease can cause up-regulation of deimination. Increased levels of deimination have been observed in photoreceptors and in the retinal pigment epithelium when animals are subjected to prolonged exposure to light (unpublished observations). Retinal photoreceptor layers in mice (CD1, C57BL6J and DBA/2J) and rats (Wistar, Sprague-Dawley and F344BN) also show diurnal variation in deimination level (unpublished observations). Ocular deimination has been found to be associated with neurodegenerative diseases such as experimental autoimmune encephalomyelitis and glaucoma4, 23, 32.
Whether protein deimination is associated with the process of aging, a specific phenotype of aging, or is likely to be mechanistically related to a disease process25 remains to be elucidated. Investigation of deimination status during the normal aging process was performed using the F1 hybrid between Fischer 344 and Brown Norway rats (F344BN), an animal model widely used in aging studies25.
In the retina of young F344BN rats (approximately 3month-old) decreased deimination has been shown in comparison with aged rats (approximately 24 month-old) (Fig. 2). Immunohistochemistry of cryosections shows that the RGC layer undergoes significant loss of deimination (Figs. 2A and 2B). Similar findings were also recorded in the aged animals when compared with the young ones25. The decrease was significant in the RGC layer, inner plexiform layer, and inner nuclear layer (Fig. 2). The observed decreased deimination in F344BN retina is commensurate with decreased PAD2 mRNA levels, activity (Fig. 3) and expression25. The loss of RGCs, retinal degeneration and optic nerve damage at corresponding advanced ages in F344BN rats is less pronounced when compared with Sprague-Dawley or Long-Evans RCS rats26. However, when accounting for the loss of cells using a ganglion cell marker, Thy-127, and a microtubule-associated protein 2 (MAP2), a marker for ganglion cell and inner plexiform layer28, 29, the normalized level of PAD2 and deimination still showed a decrease in aged F344BN when compared with young animals. In other words, in aging retina, in addition to cell loss, there was also an actual loss of deimination in residual cells. Aging studies with F344BN rats25 suggest that the elevated PAD2 and deiminated protein levels in late onset and progressive ocular diseases such as glaucoma, multiple sclerosis and experimental autoimmune encephalomyelitis4, 30 are likely due to the pathological process of disease and not age associated changes. Elevated deimination in the retina with prolonged light exposure suggests that deimination may be potentially involved in light exposure related changes and damage to the retina. PAD2 was identified using the unbiased approach of proteomic mass spectrometry in glaucomatous optic nerve but not in the control. Elevated deimination was also found in glaucomatous optic nerve, associated with elevation in intraocular pressure. Astrocytes were found to be contributory to elevated PAD2 and deimination levels4, 30.
DEIMINATION AND OTHER DISEASES
Although much of the biological consequences of deimination remain to be understood, elevated deimination and PAD2 expression have been found in several human neurological diseases such as multiple sclerosis31, 33, 34, autoimmune encephalomyelitis23, Alzheimer's35–37, amyotrophic lateral sclerosis38 and glaucoma4, 30. Elevated deimination has also been observed in other diseases such as cutaneous disorders, rheumatoid and autoimmune arthritis8, 39, 40 and scrapie-infected brain41. Deimination have been found to alter the biological activities of CXCL8/interleukin-8 (IL-8). CXCL8 is a neutrophil chemoattractant and an angiogenic cytokine. Arginine residue in position 5 of CXCL8 have been found undergo deimination. Deimination of CXCL8 by PAD alters receptor usage (reduced receptor CXCR2-dependent calcium signaling), prevents proteolysis and dampens tissue inflammation32.
The physiological role of deimination, including a possible role in neuronal remodeling and plasticity, remain to be elucidated. Rigorous determinations of substrate specificity of PADs also remain to be performed. Only further investigation can unravel the exact role and consequences of loss of deimination during aging and disease process. Current investigations suggest that in aging and disease two different set of cells undergo modulation in deimination. In normal aging, the RGCs show a decrease in deimination25 (Fig. 4A). However, in the retina of neurodegenerative diseases, such as glaucoma, the loss of deimination in the RGC layer is either small or much less apparent. Astrocytes or astroglial cells appear to undergo an elevation in deiminase levels in diseases like glaucoma4, 30. Our current hypothesis therefore is that in normal aging and neurodegenerative diseases different sets of cells undergo elevation or decrease in deimination (Fig. 4). Identification of proteins and their deimination states will enable advancement in understanding the role deimination plays in these cell types and consequences of deimination in normal biology. Future identification of specifically modified proteins will enhance our understanding about the biological role and functional pathways affected by protein deimination in the aging retina. The consequences of loss of deimination during normal aging and aberrant PAD2 activity in the pathogenic states is expected to shed new light into the biology of deimination.
We thank and acknowledge all our colleagues who participated in the original studies and Dr. Vera Bonilha for the retinal images and critical reading of the article. The original work embodied in this review was supported by the grants from Hope for Vision award, Thomas R. Lee Award from the American Health Assistance Foundation, and a RPB Career Development Award.