Murine retinal citrullination declines with age and is mainly dependent on peptidyl arginine deiminase 4 (PAD4)

T. J. Hollingsworth, Marko Radic, Sarka Beranova, Francesco Giorgianni, Yanming Wang, Alessandro Iannaccone

Research output: Contribution to journalArticle

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Abstract

PURPOSE. Citrullination is a post-translational modification (PTM) that serves many normal physiological functions. Studies have shown that this PTM—along with expression of the catalyzing enzymes, peptidyl arginine deiminases (PADs)—are increased in autoimmune and age-related pathologies. PAD2 retinal expression has been previously documented in rat and human. Herein, we report on the expression levels and patterns of PAD2, PAD4, and retinal citrullination in the murine retina with age. M ETHODS . Wild-type (WT) and Pad4 -/- (PAD4KO) mice ages 0.5, 0.75, 1, 3, 6, and 9 months were investigated after euthanasia and eye enucleation. Retinal lysates from 3-month-old mice were probed for PAD4 by western blot. Whole eyes were fixed, cryosectioned, and probed using an anti-PAD2/4 antibody (Ab), a specific anti-PAD4 Ab, and F95 anti-citrullinated peptide Ab. Fluorescent intensities were quantified with ImageJ. RESULTS. WT retinas show different levels of PAD4 expression in distinct retinal layers, with the most intense labeling in inner retinal layers, while PAD4KO mice lacked retinal PAD4. Using a nonspecific anti-PAD2/4 Ab, PAD reactivity observed in PAD4KO mice was attributed to PAD2. In WT, both PAD2 and PAD4 expression levels decrease significantly with age while low-level residual PAD2 expression was seen in PAD4KO mice. Citrullination levels in WT retinas paralleled PAD4 expression, with PAD4KO mice exhibiting consistently minimal citrullination. CONCLUSIONS. Both PAD2 and PAD4 expression and citrullination decrease with age in the murine retina. However, in the absence of PAD4, retinal citrullination is nearly abolished, indicating that PAD4 is a main effector for retinal citrullination under physiological conditions.

Original languageEnglish (US)
Pages (from-to)3808-3815
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number10
DOIs
StatePublished - Aug 1 2018

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Retina
arginine deiminase
Antibodies
Eye Enucleation
Retinaldehyde
Euthanasia
Post Translational Protein Processing
Anti-Idiotypic Antibodies
Western Blotting
Pathology
Peptides
Enzymes

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Murine retinal citrullination declines with age and is mainly dependent on peptidyl arginine deiminase 4 (PAD4). / Hollingsworth, T. J.; Radic, Marko; Beranova, Sarka; Giorgianni, Francesco; Wang, Yanming; Iannaccone, Alessandro.

In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 10, 01.08.2018, p. 3808-3815.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. Citrullination is a post-translational modification (PTM) that serves many normal physiological functions. Studies have shown that this PTM—along with expression of the catalyzing enzymes, peptidyl arginine deiminases (PADs)—are increased in autoimmune and age-related pathologies. PAD2 retinal expression has been previously documented in rat and human. Herein, we report on the expression levels and patterns of PAD2, PAD4, and retinal citrullination in the murine retina with age. M ETHODS . Wild-type (WT) and Pad4 -/- (PAD4KO) mice ages 0.5, 0.75, 1, 3, 6, and 9 months were investigated after euthanasia and eye enucleation. Retinal lysates from 3-month-old mice were probed for PAD4 by western blot. Whole eyes were fixed, cryosectioned, and probed using an anti-PAD2/4 antibody (Ab), a specific anti-PAD4 Ab, and F95 anti-citrullinated peptide Ab. Fluorescent intensities were quantified with ImageJ. RESULTS. WT retinas show different levels of PAD4 expression in distinct retinal layers, with the most intense labeling in inner retinal layers, while PAD4KO mice lacked retinal PAD4. Using a nonspecific anti-PAD2/4 Ab, PAD reactivity observed in PAD4KO mice was attributed to PAD2. In WT, both PAD2 and PAD4 expression levels decrease significantly with age while low-level residual PAD2 expression was seen in PAD4KO mice. Citrullination levels in WT retinas paralleled PAD4 expression, with PAD4KO mice exhibiting consistently minimal citrullination. CONCLUSIONS. Both PAD2 and PAD4 expression and citrullination decrease with age in the murine retina. However, in the absence of PAD4, retinal citrullination is nearly abolished, indicating that PAD4 is a main effector for retinal citrullination under physiological conditions.",
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T1 - Murine retinal citrullination declines with age and is mainly dependent on peptidyl arginine deiminase 4 (PAD4)

AU - Hollingsworth, T. J.

AU - Radic, Marko

AU - Beranova, Sarka

AU - Giorgianni, Francesco

AU - Wang, Yanming

AU - Iannaccone, Alessandro

PY - 2018/8/1

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N2 - PURPOSE. Citrullination is a post-translational modification (PTM) that serves many normal physiological functions. Studies have shown that this PTM—along with expression of the catalyzing enzymes, peptidyl arginine deiminases (PADs)—are increased in autoimmune and age-related pathologies. PAD2 retinal expression has been previously documented in rat and human. Herein, we report on the expression levels and patterns of PAD2, PAD4, and retinal citrullination in the murine retina with age. M ETHODS . Wild-type (WT) and Pad4 -/- (PAD4KO) mice ages 0.5, 0.75, 1, 3, 6, and 9 months were investigated after euthanasia and eye enucleation. Retinal lysates from 3-month-old mice were probed for PAD4 by western blot. Whole eyes were fixed, cryosectioned, and probed using an anti-PAD2/4 antibody (Ab), a specific anti-PAD4 Ab, and F95 anti-citrullinated peptide Ab. Fluorescent intensities were quantified with ImageJ. RESULTS. WT retinas show different levels of PAD4 expression in distinct retinal layers, with the most intense labeling in inner retinal layers, while PAD4KO mice lacked retinal PAD4. Using a nonspecific anti-PAD2/4 Ab, PAD reactivity observed in PAD4KO mice was attributed to PAD2. In WT, both PAD2 and PAD4 expression levels decrease significantly with age while low-level residual PAD2 expression was seen in PAD4KO mice. Citrullination levels in WT retinas paralleled PAD4 expression, with PAD4KO mice exhibiting consistently minimal citrullination. CONCLUSIONS. Both PAD2 and PAD4 expression and citrullination decrease with age in the murine retina. However, in the absence of PAD4, retinal citrullination is nearly abolished, indicating that PAD4 is a main effector for retinal citrullination under physiological conditions.

AB - PURPOSE. Citrullination is a post-translational modification (PTM) that serves many normal physiological functions. Studies have shown that this PTM—along with expression of the catalyzing enzymes, peptidyl arginine deiminases (PADs)—are increased in autoimmune and age-related pathologies. PAD2 retinal expression has been previously documented in rat and human. Herein, we report on the expression levels and patterns of PAD2, PAD4, and retinal citrullination in the murine retina with age. M ETHODS . Wild-type (WT) and Pad4 -/- (PAD4KO) mice ages 0.5, 0.75, 1, 3, 6, and 9 months were investigated after euthanasia and eye enucleation. Retinal lysates from 3-month-old mice were probed for PAD4 by western blot. Whole eyes were fixed, cryosectioned, and probed using an anti-PAD2/4 antibody (Ab), a specific anti-PAD4 Ab, and F95 anti-citrullinated peptide Ab. Fluorescent intensities were quantified with ImageJ. RESULTS. WT retinas show different levels of PAD4 expression in distinct retinal layers, with the most intense labeling in inner retinal layers, while PAD4KO mice lacked retinal PAD4. Using a nonspecific anti-PAD2/4 Ab, PAD reactivity observed in PAD4KO mice was attributed to PAD2. In WT, both PAD2 and PAD4 expression levels decrease significantly with age while low-level residual PAD2 expression was seen in PAD4KO mice. Citrullination levels in WT retinas paralleled PAD4 expression, with PAD4KO mice exhibiting consistently minimal citrullination. CONCLUSIONS. Both PAD2 and PAD4 expression and citrullination decrease with age in the murine retina. However, in the absence of PAD4, retinal citrullination is nearly abolished, indicating that PAD4 is a main effector for retinal citrullination under physiological conditions.

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