Abstract
Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, α-tubulin, and β-casein. Given the importance of prolactin to lactation, we next tested if it stimulates PAD-catalyzed citrullination using mouse mammary epithelial CID-9 cells. Stimulation of CID-9 cells with 5 µg/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in α-tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes β-casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased β-casein and butyrophilin mRNA expression; however, this increase was significantly inhibited by the pan-PAD inhibitor, BB-Cl-amidine (BB-ClA). We also examined the effect of tubulin citrullination on the overall polymerization rate of microtubules. Our results show that citrullinated tubulin had a higher maximum overall polymerization rate. Our work suggests that protein citrullination is an important PTM that regulates gene expression and microtubule dynamics in mammary epithelial cells.
Highlights
Multiple peptidylarginine deiminase (PAD or PADI) isoforms are expressed in the mammary glands of mice, dogs, cats, and humans, primarily in luminal epithelial cells [1,2,3]
Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), we identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, α-tubulin, and β-casein
Since prolactin is critical for mammary secretory cell function during lactation, we tested if prolactin stimulates citrullination of histone H2A and α-tubulin using the mouse mammary epithelial CID-9 cell line
Summary
Multiple peptidylarginine deiminase (PAD or PADI) isoforms are expressed in the mammary glands of mice, dogs, cats, and humans, primarily in luminal epithelial cells [1,2,3]. PADs are expressed in lactating mammary gland secretory cells. The small triacylglycerol-rich lipids form larger cytoplasmic lipid droplets (CLDs) that are transported to the apical membrane of mammary secretory cells. Microtubules are necessary to transport milk protein vesicles to the plasma membrane, and their disruption leads to accumulation of casein-containing vesicles in mammary secretory cells [18,24]. Since prolactin is critical for mammary secretory cell function during lactation, we tested if prolactin stimulates citrullination of histone H2A and α-tubulin using the mouse mammary epithelial CID-9 cell line. In terms of functional consequences, citrullination regulated expression of lactation genes Csn and Btn1a and altered the overall polymerization rates of microtubules, suggesting a role for this PTM in both the synthesis and secretion of milk. Our work was the first to show that β-casein was citrullinated in milk from mice and humans, raising additional interesting questions regarding the biological significance of this understudied PTM
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