Structural Analysis and protein modeling:
To model the newly identified mutation (p.122insT), the mutated CTSC amino acid sequence was compared to template databases using the online server Swiss-Prot Template Identification tool(Arnold et al., 2006; Biasini et al., 2014; Kiefer, Arnold, Künzli, Bordoli, & Schwede, 2008) on http://swissmodel.expasy.org.
The most similar template to the target mutated protein, with 100% sequence identity, was human cathepsin C, which has been solved to 2.15 Å 1K3BA including the amino acids of the first chain of the protein(Kiefer et al., 2008). Other structures with more sequence coverage in terms of the number of the amino acids than 1K3BA structure were also found, but the X-ray structure of the 1K3BA was selected for two reasons; Firstly, the complete sequence identity with the mutated region, and secondly, its better quality for the purpose of the homology modeling that can exactly describe the structural defects caused by the mutation.
The selected 1K3BA structure was submitted for homology modeling using the online Swiss-Prot server for automated modeling on http://swissmodel.expasy.org(Biasini et al., 2014; Guex & Peitsch, 1997; Schwede, Kopp, Guex, & Peitsch, 2003). The result was set for the Energy Minimization job using ZMM software. The ZMM uses the Amber all-atom force field with a cut-off distance of 10 Å and Monte Carlo Minimization Method to minimize conformational energy in the space of generalized coordinates including torsions and bond angles(Zhenqin Li & Scheraga, 1987; Zdobnov & Apweiler, 2001). The Energy Minimization was terminated after 100 sequential minimizations failed to improve the lowest-energy conformation.
The essential accuracy and correctness of the model was then evaluated using the PROCHECK and WHAT-IF programs from the online server at http://nihserver.mbi.ucla.edu/SAVES/(Vriend, 1990).
The electrostatic potential of the molecule was computed using Coulomb’s Law and the Swiss-PdbViewer 4.02, as well as the graphical representations presented here(Guex & Peitsch, 1997).
Results
A six year old boy that was diagnosed as Papillon–Lefevre syndrome affected (according to the established criteria by Gorlin et al.) was referred to Avicenna Research Institute with chief complain of a long history of hyperkeratosis and periodontitis. The majority of his deciduous teeth fell off by the age of 6 years. The family history revealed that he was born from a relative parents and the only affected one of the family(Guex & Peitsch, 1997; Zhiming Li et al., 2014). His cousin shows clinical symptoms of the disease but his siblings seem normal at the time of study (Fig 1a, 1b and 1c). The complete analysis of coding sequences and splice sites of CTSC gene revealed a novel three nucleotides GAC insertion (p.122insT) in the third exon of the gene in-patient in homozygous form, which lead to the insertion of an additional threonine in polypeptide chain. Further genotype analyses of parents showed the existence of mentioned mutation in heterozygous form (Fig 2). All the healthy control volunteers showed the wild type sequence of the gene.
The tertiary structures of the normal and mutated proteins are shown in Fig. 3. The model was also analyzed in terms of stereo-chemical and geometrical parameters such as G-Factor, bond length, and bond angles, for which the results be satisfied with the discussed criteria. In addition, most of the residues were inside the favorable regions of the Ramachandran map. After energy minimization, the overall energy of the model was -666.19 kcal/mol for the 1K3BA structure.
Structural changes that have been emerged by the insertion of the Threonine amino acid in the normal gene (p.122ins.T), as predicted from molecular modeling, are compatible with the observed phenotype.
Discussion
Although PLS is a rare disease, its psychological and social impacts on growing affected children can influence the quality of their life. The exact etiology of PLS is still ambiguous. However, Anatomy, microorganisms, immunologic response and genetic factors are responsible for development of the syndrome. Some microbial agents including Actinobacillus actinomycetemcomitans was reported as a significant factor in development of periodontal involvements(Stabholz, Taichman, & Soskolne, 1995). Recent investigations have demonstrated that PLS is mostly seen in consanguineous marriages, which is directly related to the cathepsin C abnormality. Dipeptidyl peptidase I (DPPI) or cathepsin C has a vital role in defense against pathogenic organisms by physiological activating of some serine proteases in immune cells. Therefore, deficiency of cathepsin C function will be resulted in lack of immunological response, leading to increased risk of severe infections(Basapogu Sreeramulu et al., 2015). The CTSC gene that code cathepsin C, is commonly expressed in epithelial cells and causes different clinical symptoms such as severe gingivostomatitis and periodontitis. Early diagnosis of PLS has important role in management of patient’s oral conditions.
In this study, we report a novel mutation in CTSC gene, which can lead to production of an abnormal protein according to the obtained results of DNA genomic sequencing and in Silico studies.
Genetic testing of studied family revealed presence of the novel insertion in homozygous form in the third exon of CTSC gene (p.122ins.T) of affected patient. Up till now 85% of PLS patients are in homozygous form in CTSC mutations which 13% are located in exon 1 to 3, located in the exclusion domain of the related protein (Nagy et al., 2014). Coding sequence of cathepsin c contains 463 amino acids including a signal peptide with 24 amino acids, an exclusion domain with 233 amino acids and a propeptide with 206 amino acids that it also contains the heavy and light chain regions(Nitta et al., 2005). The C-terminal portion of the exclusion domain is a conserved region, which is thought to be important for enzyme activity. Mutation in this region blocks access to the active site and inhibits enzyme to bind substrates.
Structural analyses of the mutated protein also revealed important changes in its tertiary structure due to the elucidated three nucleotides insertion (p.122ins.T) in the active site of Dipeptidyl peptidase I domain (cathepsin C), exclusion domain, SSF75001, accessed by InterproScan(Zdobnov & Apweiler, 2001). As in Figure 3a, which is related to the normal protein model, the amino acids threonine, methionine, and threonine are at positions 121, 122, and 123 in exclusion domain of the protein, respectively, but after the mutation (p.122ins.T), a threonine after threonine 121 is added. Proximity of three threonines can also alter the protein structure, leading to poor or inappropriate protein function; this change is shown in figure 3b. Therefore, due to the strong relationships between the protein tertiary structure and its functions, it can be realized that such important abnormality in the structure of the mutated protein can be cause consequent important abnormalities in the protein function.
In conclusion, according to the results obtained from this study, we can justify correlation between this reported mutation and observed phenotype in this 6-year-old-boy affected by Papillon–Lefevre syndrome.
Acknowledgment
This work was supported by a grant from the Vice Chancellor for Research at Mashhad University of Medical Sciences, no. 910523.
Conflict of interest
The authors declare no potential conflicts of interest.
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