Abstract
The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren's syndrome.
Highlights
In certain autoimmune diseases, components of the immune response recognize epitopes at peptidylarginine to peptidylcitrulline posttranslationally modified residues
All experimental procedures with animals were performed according to the Mexican Guidelines for the Production, Care, and Use of Laboratory Animals (NOM-062-ZOO-1999) and the International Guide for the Care and Use of Laboratory Animals, and experiments were performed according to the guidelines for ethical conduct in the care and use of animals developed by the American Psychological Association (APA)
The main findings suggest that peptidylarginine deiminases (PADs) activity may be induced in salivary glands in tissue culture by different triggers and that the induced PAD enzymes are capable of stereochemically modifying the arginine residues of Ro60 and La ribonucleoproteins
Summary
Components of the immune response recognize epitopes at peptidylarginine to peptidylcitrulline posttranslationally modified residues. This catalytic pathway involves the activity of peptidylarginine deiminases (PADs), which induce a hydrolytic reaction in arginine that results in protein citrullination. This stereochemical process modifies the physicochemical behaviour of the target protein by increasing its antigenicity [1]. PADs are involved in basic processes of cell physiology such as fertilization and embryo development; the citrullination of histones by PAD4 antagonize the histone methylation induced by the histone arginine methyltransferase and affect the gene expression [3,4,5]. PADs participate in the terminal differentiation of the skin [6]
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