Abstract
In eukaryotes, cellular functions are tightly controlled by diverse post-translational modifications (PTMs) of proteins. One such PTM affecting many proteins is the deimination of arginine to citrulline. This process, called citrullination is catalyzed by a group of hydrolases called protein arginine deiminases (PADs), of which five isoforms have been identified. Hypercitrullination, as a result of increased PAD expression or activity, is associated with autoimmune diseases e.g., rheumatoid arthritis, lupus, Alzheimer’s disease, ulcerative colitis, multiple sclerosis, and certain cancers. Three common single nucleotide polymorphisms (SNPs) in the PADI4 gene have been described, namely rs874881, rs11203366, and rs11203367, which are thought to affect PAD4 expression and activity. We here compared the suitability of four methods for the screening of SNPs in the PADI4 gene: (i) SYBR-green based real-time polymerase chain reaction followed by high resolution melting curve analysis (HRM-PCR); (ii) PCR followed by detection of restriction fragment length polymorphisms (PCR-RFLP); (iii) conventional tetra-primer amplification refractory mutation system PCR (ARMS-PCR); and (iv) real-time PCR based on the one-step ARMS-PCR. Of these, ARMS-PCR proved to be the most suitable method regarding handling, duration, and cost of experiments. Using the method with SYBR-green based real-time PCR reagents further diminished handling steps and thus potential sources of error.
Highlights
In eukaryotes over 200 different post-translational modifications (PTMs) of proteins are described
Phosphorylation, ADP-ribosylation, and citrullination [1,2,3]. The latter is catalyzed by a small family of hydrolases called protein arginine deiminases (PADs)
PAD4 being expressed in bone marrow, CD34+ cells, granulocytes, lymphocytes, monocytes, and macrophages, targets mainly histones H3 and H4, as well as the stress response gene OKL38 [7]
Summary
In eukaryotes over 200 different post-translational modifications (PTMs) of proteins are described. Phosphorylation, ADP-ribosylation, and citrullination [1,2,3]. The latter is catalyzed by a small family of hydrolases called protein arginine deiminases (PADs). The tissue and substrate specificity of the PADs define their role in health and disease [5,6]: PAD1, which targets keratins, has been detected in epidermis, keratinocytes, sweat glands, and growing hair follicles. PAD2, targeting a broader spectrum of proteins, including histone H3, vimentin, enolase, and tubulin, is reported to be expressed in epidermis, keratinocytes, lymphocytes, monocytes, macrophages, neutrophils, oligodendrocytes, and Schwann cells. PAD3, which targets keratins and S100A3, is expressed in epidermis, keratinocytes, growing hair follicles, neutrophils, and Schwann cells. PAD4 being expressed in bone marrow, CD34+ cells, granulocytes, lymphocytes, monocytes, and macrophages, targets mainly histones H3 and H4, as well as the stress response gene OKL38 [7]
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