Identification and Characterization of the Lactating Mouse Mammary Gland Citrullinome

• Published in: International journal of molecular sciences Vol. 21; no. 7; p. 2634
• Main Authors: Li, Guangyuan, Young, Coleman H, Snow, Bryce, Christensen, Amanda O, Demoruelle, M Kristen, Nemmara, Venkatesh V, Thompson, Paul R, Rothfuss, Heather M, Cherrington, Brian D
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 10.04.2020; MDPI
• Subjects: Animals; Arginine; Arginine - metabolism; Breastfeeding & lactation; Butyrophilin; Casein; Cells, Cultured; citrullination; Citrullination - drug effects; Citrulline; Citrulline - metabolism; Enzymes; Epithelial cells; Female; Gas Chromatography-Mass Spectrometry; Gene Expression; Histone H2A; Histones; Histones - metabolism; Humans; Kinases; Lactation; Lipids; Mammary gland; Mammary glands; Mammary Glands, Animal - metabolism; Mass spectrometry; Mass spectroscopy; Mice; Microtubules; Milk; Milk Proteins - metabolism; Peptides; peptidylarginine deiminase; Physiology; Polymerization; Post-translation; Prolactin; Prolactin - metabolism; Prolactin - pharmacology; Protein Processing, Post-Translational; Protein-arginine deiminase; Protein-Arginine Deiminases - metabolism; Proteins; Proteome; Proteomics - methods; RNA, Messenger - genetics; Scientific imaging; Time Factors; Tubulin; prolactin; mammary gland; peptidylarginine deiminase; lactation; citrullination
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21072634

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 ( ) and butyrophilin ( ). 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.