Purification of enzymatically inactive peptidylarginine deiminase type 6 from mouse ovary that reveals hexameric structure different from other dimeric isoforms

Abstract

The murine peptidylarginine deiminase (PAD) has five isoforms encoded by different genes and participates in a variety of cellular functions through the citrullination of target proteins. The crystal structure of human PAD4 with a dimeric form was previously solved because of the enzyme’s relevance to rheumatoid arthritis. PAD6, abundant in mouse oocytes and eggs, is believed to take part in early events of embryogenesis, but its biochemical properties are little understood. Here we have purified and characterized a recombinant PAD6. A PAD6 cDNA was cloned from mouse ovary RNA and expressed in Escherichia coli through pET29 and pGEX vectors. When benzoyl-L-arginine ethyl ester was used as a substrate, no appreciable activity was detected with a cell homogenate under conditions where a human PAD4 cDNA caused significant activity. Both proteins were affinity-purified to near homogeneity. The circular dichroism spectra of PAD6 and human PAD4 were similar in the far ultraviolet region. On molecular sieving, PAD6 was eluted faster than human PAD4. The cross-linking of PAD6 with dimethyl suberimidate clearly showed six bands on an sodium dodecyl sulfate-polyacrylamide gel. These results indicate that PAD6 can constitute a hexameric structure. The purified PAD6 still showed no enzymatic activity. This unique structure and loss in enzymatic activity is strongly suggested to favor the formation of egg cytoplasmic sheets as the architectural protein

Mining the Human Tissue Proteome for Protein Citrullination

“…This irreversible PTM leads to a small increase in mass of 0.9840 Da and the loss of a positive charge from the protein which may cause structural and/or functional alterations. Five PAD isoforms have been found in humans, PAD1-4, and the catalytically inactive PAD6 (1), which are believed to have distinct tissue-specificities (2-6). PADcatalyzed citrullination has been implicated in many cellular processes such as terminal epidermal differentiation, apoptosis, central nervous system (CNS) stability, immune response, gene regulation, and embryonic development (7,8).…”

Development of Activity-Based Proteomic Probes for Protein Citrullination

“…There are five human PADs, i.e., PAD1, PAD2, PAD3, PAD4, and PAD6, of which only PADs 1–4 are catalytically active (Raijmakers et al 2007; Taki et al 2011; Witalison et al 2015). These isozymes, which share 50–55% sequence identity within the same species (Arita et al 2004), are found in a myriad of cells and tissue types.…”

“…This PTM is generated by a family of calcium-dependent enzymes called protein arginine deiminases (PADs) (Bicker and Thompson 2013; Fuhrmann et al 2015; Fuhrmann and Thompson 2016). There are five human PADs, i.e., PAD1, PAD2, PAD3, PAD4, and PAD6, of which only PADs 1–4 are catalytically active (Raijmakers et al 2007; Taki et al 2011; Witalison et al 2015). These isozymes, which share 50–55% sequence identity within the same species (Arita et al 2004), are found in a myriad of cells and tissue types.…”

Synthesis and Biological Evaluation of an Indazole-Based Selective Protein Arginine Deiminase 4 (PAD4) Inhibitor

“…3−5 PAD1 to PAD4 are catalytically active, but PAD6 has no detectable activity. 2,5,6 Although these isozymes share greater than 50% interisozyme sequence identity, 1,5 they display unique tissue expression patterns. 4,7 Significantly, dysregulation of PAD activity has been linked to the pathogenesis of many diseases.…”

“…Citrullination is a post-translational event involved in many cellular processes such as gene regulation and cell differentiation. , This post-translational modification results in the loss of the net positive charge on the target protein, which leads to structural changes of the protein and consequently alters the protein function and its interaction with other biomolecules. Five human PADs (1, 2, 3, 4, and 6) have been identified and characterized. − PAD1 to PAD4 are catalytically active, but PAD6 has no detectable activity. ,, Although these isozymes share greater than 50% interisozyme sequence identity, , they display unique tissue expression patterns. , Significantly, dysregulation of PAD activity has been linked to the pathogenesis of many diseases. ,, …”