Abstract
Peptidylarginine Deiminases (PADs) convert arginine residues on substrate proteins to citrulline. Previous reports have documented that PAD2 expression and activity varies across the estrous cycle in the rodent uterus and pituitary gland, however, the expression and function of PAD2 in mammary tissue has not been previously reported. To gain more insight into potential reproductive roles for PAD2, in this study we evaluated PAD2 expression and localization throughout the estrous cycle in canine mammary tissue and then identified possible PAD2 enzymatic targets. Immunohistochemical and immunofluorescence analysis found PAD2 expression is low in anestrus, limited to a distinct, yet sparse, subset of epithelial cells within ductal alveoli during estrus/early diestrus, and encompasses the entire epithelium of the mammary duct in late diestrus. At the subcellular level, PAD2 is expressed in the cytoplasm, and to a lesser extent, the nucleus of these epithelial cells. Surprisingly, stimulation of canine mammary tumor cells (CMT25) shows that EGF, but not estrogen or progesterone, upregulates PAD2 transcription and translation suggesting EGF regulation of PAD2 and possibly citrullination in vivo. To identify potential PAD2 targets, anti-pan citrulline western blots were performed and results showed that citrullination activity is limited to diestrus with histones appearing to represent major enzymatic targets. Use of site-specific anti-citrullinated histone antibodies found that the N-terminus of histone H3, but not H4, appears to be the primary target of PAD activity in mammary epithelium. This observation supports the hypothesis that PAD2 may play a regulatory role in the expression of lactation related genes via histone citrullination during diestrus.
Highlights
The peptidylarginine deiminases (PADs) are a family of calciumdependent enzymes that post-translationally convert arginine residues on substrate proteins to the non-standard amino acid citrulline
Canine Peptidylarginine Deiminase 2 (PAD2) is expressed in the mammary gland and its expression varies in an estrous cycle-dependent manner
PAD4 IHC staining of canine mammary tissue sections indicate that PAD4 staining is lowest during late diestrus supporting the hypothesis of a unique role for PAD2 during late diestrus (Figure S1B)
Summary
The peptidylarginine deiminases (PADs) are a family of calciumdependent enzymes that post-translationally convert arginine residues on substrate proteins to the non-standard amino acid citrulline. PAD catalyzed citrullination, with concomitant loss of the positive imine group, converts the strongly basic arginine residue to a neutral amino acid. Loss of basic charge caused by citrullination is thought to disrupt charge distribution within the substrate protein and alter its ability to interact with other molecules [1,2]. The PAD family consists of five members (1–4 and 6) located within a gene cluster encompassing ,300 kb at human chromosome 1p36.13. The PAD enzymes and citrullinated proteins are associated with multiple human diseases including rheumatoid arthritis, multiple sclerosis, Alzheimer’s disease, and, more recently, with cancer [4,5,6,7]
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