PRRT2 gene and protein in human: characteristics, evolution and function

The proline-rich transmembrance protein 2 (PRRT2) gene located in chromosome 16 p11.2 has 4 exons with a total length of 3794 bases and encodes 340 amino acids. The PRRT2 protein is a presynaptic membrane protein that plays an important role in cell exocytosis and neurotransmitter release. However, the detailed functions of the protein remain unclear. Chen et al. discovered for the first time the causative mutation of this gene in paroxysmal kinesigenic dyskinesias (PKD) in 2011 [1]. Subsequent studies have further confirmed that mutations in the PRRT2 gene are a major cause of PKD. In addition, the PRRT2 gene is also involved in the benign familial infantile seizures (BFIS) and infantile convulsions with paroxysmal choreoathetosis (ICCA) [2, 3]. So far, 88 different mutations of PRRT2 have been reported, including 40 missense/nonsense mutations, 22 small deletions, 15 small insertions, 5 splicing mutation, 5 gross deletions and 1 gross insertion/duplication. Among them, the c.649 mutation has been the hotspot, which contains three mutation forms c.649dupC, c.649delC and c.649C > T. The pathogenic mutations of PRRT2 have also been found in paroxysmal nonkinesigenic dyskinesias (PNKD), paroxysmal exercise-induced dyskinesias (PED), familial hemiplegic migraine (FHM), episodic ataxia (EA), febrile seizures (FS), infantile non-convulsive seizures (INCS), nocturnal convulsions (NC) and paroxysmal torticollis [4‐10]. At present, most studies have focused on the identification of phenotype profiles of PRRT2 mutations, while the specific physiological functions of PRRT2 are rarely studied.

Bioinformatics is a field of science that combines biology, computer science, engineering, and applied mathematics to process and analyze information on DNA and protein sequences and structures, based on the massively stored biological experiments and derived datasets. The bioinformatics discipline contributes to the establishment of theoretical models, the setup of experimental research, and the genomics and proteomics studies. In this study, we set out to analyze the physical and chemical properties and molecular structure of PRRT2 using the bioinformatics approach, and predict the functions of PRRT2 in cells. In addition, as the sequence of the human PRRT2 gene promoter has not been recorded in the NCBI database, and no bioinformatic analysis of the PRRT2 promoter has been reported, we also screened for potential promoter sequences of the human PRRT2 gene from the genomic database and analyzed transcription factors as well as their binding sites and CpG islands in this gene. The bioinformatics results will lay a foundation for in-depth study of functions of PRRT2 in the pathogenesis of PKD and other diseases, and for the design of gene therapy. This study will also provide theoretical reference for the construction of PRRT2 gene promoter expression vector and determination of the gene promoter function in subsequent experimental studies.

PRRT2 is a proline-rich transmembrane protein type II encoded by 3 exons (exons 2–4), with a total length of 340 amino acids. Recent studies had revealed that the long N-terminus of PRRT2 is located inside the cell, and the C-terminus, which contains only 2 residues, is located outside the cell [17]. PRRT2 is enriched in the presynaptic membrane of neurons in the cerebral regions such as the cortex, hippocampus, basal ganglia and cerebellum, and interacts with the core protein of the SNARE complex, participating in the regulation of synaptic neurotransmitter release and promoted exocytosis of vesicles. PRRT2 also plays an important role in synaptic triggering and synaptic function, and thus has been proposed to be a new synaptic protein [17].

In this study, the amino acid sequences of PRRT2 of different species were obtained from public databases. Homology analysis showed that the human PRRT2 gene and that of other mammalian species were highly conserved during evolution. PRRT2 was predicted to be an unstable hydrophilic protein located on the plasma membrane, which contained two transmembrane domains. The top 10 proteins predicted by the String database to interact with PRRT2 were involved in the Rap1 signaling pathway, the Ras signaling pathway and the MAPK signaling pathway. The promoter, located near the transcription initiation site, is a DNA sequence to which an RNA polymerase can bind to initiate transcription. Here, we used three softwares to analyze promoters within the 2000 bp 5′ upstream sequence of human PRRT2 gene based on different principles and algorithms, and found that the gene had at least two potential promoter regions in the chain of justice, and the TSS was located at 1240 bp G base. In the gene expression regulation network, the combination of transcription factors and cis-acting elements can switch on or off the expression of a specific set of genes. Here, we used AliBaba2.1 and PROMO to predict transcription factor-binding sites in the promoter region of PRRT2 gene. Thirteen transcription factors were simultaneously predicted by both softwares and at the same binding site. The probability of the existence of these transcription factors was relatively high. These predictions provided evidence for the functions of PRRT2, and suggested that PRRT2 expression was regulated by a variety of transcription factors and that PRRT2, which was in a complex metabolic network, had many important physiological functions. Methylation of the CpG island can inhibit the normal transcription process of the promoter, thereby reducing gene expression. In this study, the EMBOSS and MethPrimer softwares predicted consistently the CpG islands in the promoter region of PRRT2 gene. There was one CpG island in the promoter region of human PRRT2, which was located between 1642 bp–1945 bp of [18] the 2000 bp sequence in the 5 ‘regulatory region, close to the first exon, consistent with the distribution characteristics of CpG island. Some studies have proposed that the transcriptional repression of promoter methylation could hinder the recognition of the binding site by transcription factors, thereby exerting transcription repression [19]. Sp1 is a zinc finger structural protein belonging to the transcription factor SP family, whose classical binding sites are rich in CpG sites [20]. We speculated that Sp1 may directly bind to the promoter region of PRRT2 as a transcription factor, change the promoter activity, and then regulate transcription. It has been reported that methylation of the promoter region can block the binding of transcription factor Spl to the promoter sequence and inhibit the transcription of target genes [21, 22]. Studies have confirmed that PRRT2 can interact with the SNARE complex component synaptosomal-associated protein (SNAP25) and is co-localized in the presynaptic and postsynaptic membranes [18, 23]. Subsequent evidence has supported the localization of PRRT2 in the presynaptic membrane, especially enriched in the synaptic junctions [17, 18, 24, 25]. However, the exact physiological role of PRRT2 in the presynaptic membrane remains unclear. Valente et al. have confirmed that PRRT2 is a presynaptic membrane protein that is enriched in presynaptic terminals and expressed upon the occurrence of embryonic synapses [17]. The clinical phenotypes caused by PRRT2 mutation vary broadly, including a variety of episodic phenotypes from dyskinesia to epilepsy. Even the same mutation (c.649dupC) can result in different phenotypes, such as PKD, ICCA, benign familial infantile epilepsy, and FS. These results indicated that the PRRT2 gene has the same pleiotropic characteristics as GluT1 and ATP1A2 genes [26‐28]. PRRT2 protein is widely expressed in the nervous system, particularly in the globus pallidus, cerebellum, subthalamic nucleus, cerebellar foot, caudate nucleus, cerebral cortex, hippocampus, and cerebellum [29]. Studies have confirmed that the mRNA level of PRRT2 changes with the development of mouse brain. The PRRT2 mRNA began to be expressed on embryonic day 16 and then gradually increased. By the 7th day after birth, it is expressed in the brain and spinal cord, and by the 14th day after birth (corresponding to 1–2 years in humans), the mRNA level of PRRT2 reached its peak, and then declined to a relatively low level in adult mice [29]. Moreover, the change of PRRT2 expression with age is consistent with the pathogenesis of some PRRT2-related diseases, such as the age-dependent characteristics of BFIS [30]. Therefore, the high expression of PRRT2 in the brain and the age-dependent expression pattern can partially explain the heterogeneity of PRRT2 mutation-related phenotypes.

PRRT2 mutations are associated with a variety of sudden diseases such as dyskinesia, epilepsy and migraine, indicating an overlap between the molecular pathogenesis of these diseases. It had been confirmed that PRRT2 proteins are mainly expressed in the cerebral cortex, hippocampus, basal ganglia and cerebellum, and enriched in the presynaptic membrane of neurons. More importantly, these areas are in line with the neuronal origin of putative PRRT2-related diseases. In the PRRT2-related diseases, the heterogeneous distribution of PRRT2-positive excitatory neurons and inhibitory neurons in different brain regions, and the insufficient single dose of PRRT2 caused by mutations, may lead to region-specific neuronal excitability and inhibitory. The imbalance between them can eventually lead to synaptic deregulation and neuronal hyperexcitability, triggering the onset of episodic diseases such as dyskinesia, epilepsy and migraine.

In this study, we first obtained PRRT2 gene sequences from the NCBI GenBank database, obtained the 2000 bp sequence upstream to the 5′ flanking of the PRRT2 gene, and then used different bioinformatics softwares to predict the promoter, CpG island and transcription factors of the PRRT2 gene. The results provided a basic theoretical basis for the construction of vectors for PRRT2 gene promoter expression and the detection of promoter activity, and the in silico data can provide reference for future functional studies. However, more studies are needed to advance the research on PRRT2.