Open Life Sci. 2017; 12: 248–265 Research Article Galina Laputková*, Mária Bencková, Michal Alexovič, Vladimíra Schwartzová, Ivan Talian, Ján Sabo Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment https://doi.org/10.1515/biol-2017-0030 revealed information about potential risk factors Received June 13, 2017; accepted July 24, 2017 associated with the development of caries-susceptibility and provides a better understanding of tooth protection Abstract: Background: Dental caries disease is a dynamic mechanisms. process with a multi-factorial etiology. It is manifested by demineralization of enamel followed by damage Keywords: saliva; proteins; caries-susceptible; caries- spreading into the tooth inner structure. Successful free; proteomic analysis; bioinformatics early diagnosis could identify caries-risk and improve dental screening, providing a baseline for evaluating personalized dental treatment and prevention strategies. Methodology: Salivary proteome of the whole 1 Introduction unstimulated saliva (WUS) samples was assessed in Dental caries, resulting in demineralization of the tooth caries-free and caries-susceptible individuals of older structure, is ranked among the most prevalent chronic adolescent age with permanent dentition using a diseases of people worldwide [1-3]. Although it is not a life- nano-HPLC and MALDI-TOF/TOF mass spectrometry. threatening disorder, it still represents a serious health Results: 554 proteins in the caries-free and 695 proteins in issue with a significant effect on general health and the caries-susceptible group were identified. Assessment quality of life [4,5]. using bioinformatics tools and Gene Ontology (GO) term A complex set of interactions between acid producing enrichment analysis revealed qualitative differences bacteria and fermentable carbohydrates contribute to between these two proteomes. Members of the caries- caries-risk [6,7]. Other factors associated with caries are susceptible group exhibited a branch of cytokine saliva properties [8], genetic predispositions [9,10], age binding gene products responsible for the regulation and immunological status, and behavioral factors like of immune and inflammatory responses to infections. nutrition level and hygiene habits [2,11]. Inspection of molecular functions and biological The diagnostic assessment using the molecular processes of caries-susceptible saliva samples revealed analysis (e.g., to find protein markers) related to formation significant categories predominantly related to the of tooth decay at early stages may help to identify risk activity of proteolytic peptidases, and the regulation factors and help with dental screening and personalized of metabolic and catabolic processes of carbohydrates. dental treatment. Conclusions: Proteomic analysis of the whole saliva Recently, intensive investigation of protein functions in saliva as possible indicators for predicting caries-risk has begun using the state-of-the-art methodologies such as *Corresponding author: Galina Laputková, Department of Medical metabolomics, genomics, proteomics and bioinformatics and Clinical Biophysics, Faculty of Medicine, University of P. J. [12]. Šafárik in Košice, Galina Laputková, PhD., Trieda SNP 1, Košice, 040 Vitorino et al. [13] published an early proteomic 11, Slovakia, E-mail: [email protected] analysis evaluating the proteome of human saliva and the Mária Bencková, Michal Alexovič, Ivan Talian, Ján Sabo, Department of Medical and Clinical Biophysics, Faculty of Medicine, University of level of protein expression and adsorption on the human P. J. Šafárik in Košice, Trieda SNP 1, Košice, 040 11, Slovakia dental enamel surface. The authors compared the whole Vladimíra Schwartzová, 1st Department of Stomatology, Faculty of saliva proteome of male individuals having no dental caries Medicine, University of P. J. Šafárik in Košice, Trieda SNP 1, Košice, to those afflicted with dental caries. Both saliva incubated 040 11, Slovakia with hydroxyapatite as well as in vivo extracts from the Open Access. © 2017 Galina Laputková et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivs 3.0 License. Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 249 surface implants of tooth enamel were used as samples. acid derivative of histatin-5. It was demonstrated that The analysis showed a large number of phosphopeptides hLF1-11 and P-113 display antibacterial activity against (proline rich protein (PRP) 1/3, histatin-1, and statherin) in dental cavity-inducing S. mutans through an intracellular saliva of subjects without caries. According to an in vitro mechanism that could involve DNA binding [25]. study [14], acidic PRP, histatins and statherin preferentially In the last decade it has been established that bind to hydroxyapatite. The role of the pellicle proteins, matrix metalloproteinases present in the oral cavity PRP and statherin in maintaining dental integrity by may play a role in caries susceptibility. Several matrix promoting remineralization of the enamel has been well metalloproteinases were found to have a role in tooth established [15-17]. The authors’ data analysis showed a development, the organization of enamel and dentin statistically significant correlation between the amount of organic matrix, or in regulation of mineralization by acidic PRP, lipocalin, cystatin SN and cystatin S and the controlling the proteoglycan turnover. They seem to absence of dental caries. Samples of patients with a high play a part in dentinal caries progression through dentin Decay-missing-filled teeth index (DMFT index) correlated collagen breakdown in caries lesions [26,27]. positively with high levels of amylase, immunoglobulin A A qualitative proteomic analysis to study the salivary and lactotransferrin. proteome using the WUS of older adolescent volunteers Hong et al. [18] performed a series of experiments with permanent dentition is presented. To assess the involving Streptococcus mutans (a representative oral potential risk factors connected to tooth decay progression pathogen) to find a correlation between the presence at early stages and to evaluate other functionalities of lipoteichoic acid-binding proteins (as the major and factors related to observed oral conditions, the component of the cell wall of gram-positive bacteria determined proteins were processed and classified using in the whole saliva) in caries-susceptible and caries- bioinformatics tools. The investigation of the caries-free free subjects. A total of eight lipoteichoic acid-binding group compared to caries-experienced subjects provides a proteins in saliva of subjects without caries and twelve comprehensive proteome profile and helps with searching lipoteichoic acid-binding proteins in the individuals for particular caries-susceptible agents aimed to help with with tooth decay were identified. Histone H4, profilin-1 early dental diagnoses. and neutrophil defensin-1 were found in the caries-free group, while cystatin C, cystatin SN, cystatin S, cystatin D, lysozyme C, calmodulin-like protein 3 and b-actin 2 Materials and methods were found in the caries-susceptible group. Hemoglobin subunits A and B, prolactin-inducible protein, protein 2.1 Chemicals and reagents S100-A9, and Short palate lung nasal clone 2 (SPLUNC2) were found in both groups [18]. Identified proteins such Protease Inhibitor Cocktail for use with mammalian as histone H4, profilin-1 and neutrophil defensin-1 in cell and tissue extracts was obtained from Sigma (St. subjects without caries could play a role in antimicrobial Louis, USA). DL-Dithiothreitol (DTT), urea, thiourea, host defense, while histones may contribute to destruction iodoacetamide, Tris-HCl, mineral oil, Quick Bradford of the bacterial membrane [19], and profilins serve as hubs assay kit, and bovine serum albumin were purchased that control a complex network of molecular interactions from BioRad (Hercules, USA). Acetone and calcium [20]. Profilin-1 specifically contributes to host defense chloride were purchased from AppliChem (Darmstadt, by affecting mobility of the cells in the cytosol [21]. Germany), and acetonitrile (ACN), and trifluoroacetic acid Similarly, neutrophil defensin-1 exhibits broad-spectrum (TFA) were from Merck (Darmstadt, Germany). For the antimicrobial activity by binding to specific sites on the protein digestion, a porcine trypsin suitable for protein cell membrane with subsequent release of cellular ATP sequencing was obtained from Promega (Madison, WI, in the absence of cytolysis [22,23]. The proteins identified USA). All chemicals and reagents including ethanol, in caries-susceptible subjects, like hemoglobin, protein methanol, formic acid (FA) and glycerol were of analytical S100-A9, SPLUNC2 and prolactin-inducible protein, may grade, suitable for the electrophoresis and/or for the mass contribute to the host innate immunity in the oral cavity spectrometry measurements. Digested protein extracts [18,24]. were separated on ImmobilineDryStrips (pH 3-10, 13 Huo et al. [25] studied the activity of the two protein cm) purchased from GE Healthcare Life Sciences (Little components of saliva and their possible role in the Chalfont, United Kingdom). All solutions were prepared regulation of dental caries, i.e., a fragment of active using ultra-pure water produced by MilliQ Integral 3 water lactotransferrin domain hLF1-11 and P-113 and a 12-amino purification system from Merck (Darmstadt, Germany).