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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