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; ; 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 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 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 -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, 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, 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 -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 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 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). 250 G. Laputková, et al.

2.2 Collection of saliva and sample 2.4 In-solution protein digestion preparation The protein pellet was re-suspended in the 8 mol l-1 WUS was collected from volunteers/individuals. The urea/100 mmol l-1 Tris-HCl buffer (pH=8) to obtain total inclusion criteria involved overall systemic health with protein concentration of 1 mg ml-1. The reduction of no current or recent medications. Smokers or occasional disulfide bonds was achieved by addition of 0.1 mol l-1 smokers were excluded from the study. The individuals DTT/100 mmol l-1 Tris-HCl buffer (pH=8) and incubation who fit the inclusion criteria were classified into two at 37°C for 30 min with vortexing at 750 rpm. After that, groups: a group of 19 year old volunteers with DMFT = 0 the 0.5 mol l-1 of iodoacetamide/100 mmol l-1 Tris-HCl and a caries experienced group with DMFT > 0 (i.e., buffer (pH=8) serving as alkylating reagent was added. 4–9). Volunteers were asked to give up the morning The sample was incubated in the dark at 37°C for another oral hygiene and eating 1 h prior to saliva sampling. 30 min with vortexing at 750 rpm. Afterwards, the proteins To reduce effects of circadian rhythm, saliva samples were precipitated by pre-cooled acetone. The sample were collected between 8–10 a.m. Approximately 5 ml of was incubated for 60 min at −25°C, then vortexed and saliva was collected by expectoration into sterile 50 ml centrifuged at 4000xg for 50 min at 4oC. After discarding polypropylene tubes (BD Falcon, BD Bioscience, New the supernatant, the pellet containing proteins was Jersey, USA) placed on ice. After collection, the 1 μl of dried in a vacuum concentrator. The proteins were then protease inhibitor cocktail per 1 ml of saliva was added resuspended in the 8 mol l-1 urea/100 mmol l-1 Tris-HCl to prevent the sample degradation. In the following step, buffer (pH=8) to obtain total protein concentration of supernatant containing saliva proteins and non-soluble 1 mg ml-1. The urea concentration was reduced by diluting debris, food fragments and bacterial cells were separated with 2 mmol l-1 calcium chloride/10 mmol l-1 Tris-HCl by centrifugation at 14000×g for 30 min at 4°C. The pellet buffer up to 2 mol l-1 final concentration of urea. Standard was discarded and the supernatant stored at −80°C for overnight digestion was carried out by adding 1 µl of further analysis. trypsin (0.1 µg µl-1) to 10 µg of proteins and incubated Salivary proteins were precipitated by mixing the overnight at 37°C with vortexing at 750 rpm. Trypsin supernatant with acetone and 0.2% DTT mixed in 1:5 activity was inhibited by acidification with 20% TFA. (v/v) ratio. After overnight incubation at −25oC, vortexing followed by centrifugation at 14000×g for 30 min at 4oC was carried out. The pelleted proteins were washed 2.5 Sample desalting three times with glacial acetone and dried in the vacuum concentrator (CentriVap, Labconco, Kansas City, USA). In the next step, removal of salts, ampholytes and other Informed consent: Informed consent has been possibly interfering substances was carried out using obtained from all individuals included in this study. solid phase extraction (SPE) cartridges − BondElut -1 Ethical approval: The research related to human C18 50 mg ml (Agilent Technologies, Santa Clara, CA, use has been complied with all the relevant national USA). Initially, the C18 column was activated by double regulations, institutional policies and in accordance washing with both the 1 ml ACN:H2O (50:50, v/v) and the the tenets of the Helsinki Declaration, and has been 1 ml H2O:ACN:TFA (94.5:5:0.5, v/v/v). The 1 ml of sample approved by the Ethics Committee of the Medical Faculty, solution was then loaded onto the SPE column. At first,

University of P. J. Šafárik in Košice (protocol number: unbound components were washed off by H2O:ACN:TFA 2N/2017). (94.5:5:0.5, v/v/v). After that, the peptides were eluted

from the column with 1 ml ACN:H2O:FA (70:29.9:0.1, v/v/v) to low adhesion tubes. The sample volume was reduced 2.3 Total protein concentration assay and the ACN removed by vacuum concentration.

A Bradford’s assay (Quick Start Bradford Protein Assay, BioRad, Hercules, California, USA) with bovine gamma 2.6 Off-gel fractionation albumin was used for determining the total level of protein in the solution. Absorption was measured at In the next stage, the saliva peptides were subjected to 595 nm of wavelength using UV-Vis spectrophotometer electro-migration separation using Agilent 3100 OFFGEL (UV-3600 Spectrophotometer, Shimadzu corp., Kyoto, Fractionator (Agilent Technologies, Santa Clara, CA, USA). Japan). The sample was diluted with peptide OFFGEL solution Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 251 containing glycerol and IPG buffer (pH 3-10). The 0.15 ml 2.9 Protein database search aliquots were injected per well. A 1 mg of saliva peptides were loaded onto each IPG strip and then separated into MS a MS/MS spectra were searched by the MASCOT 12 fractions in a pH 3-10. The separation was performed 2.4 search engine (Matrix Science Ltd., UK) against the according to default standard peptide protocol: OG12PE00. SwissProt database (December, 2015). Database search Collected fractions were desalted on SPE cartridges once parameters were: taxonomy Homo sapiens (human), again and concentrated under vacuum near to dryness. fixed modification Carbamidomethylation (C), variable modification Oxidation (M), Trypsin, number of maximum missed cleavages 2, mass error tolerance 100 2.7 Nano-HPLC analysis ppm for MS spectra, 0.5 Da for MS/MS spectra and false discovery rate (FDR) ˂ 1. Peptides were separated by nano-HPLC system (UltiMate 3000, Dionex, Germany). The trap column (Acclaim PepMap 100, 100 μm×20 mm, 5 μm, 100 Å, Thermo 3 Results and discussion Scientific) and analytical column (Acclaim PepMap RSLC, As it is widely debated among researchers whether there 75 μm×15 cm, 2 μm, 100 Å, Thermo Scientific) were used is a significant difference in the protein profile between as stationary phases for preconcentration and separation, caries-free and caries-susceptible individuals, we respectively. The mobile phase consisted of 0.1% FA + 98% therefore analyzed a proteome of saliva samples of older water + 2% ACN (v/v/v, solution A) and 0.1% FA + 95% adolescent aged volunteers with permanent dentition in ACN + 5% H O (v/v/v, solution B) operated at a constant 2 this study. To assess the potential risk factors connected to flow rate of 300 nl min-1 during 150 min run-time in the dental caries, the volunteers were divided into two groups following gradient profile: 10 min at 4% B, 120 min at according to caries status. 4–40% B, 1 min at 40–95% B, 5 min at 95% B, 1 min at By using the Mascot database (excluding common 95–4% B and 13 min at 4% B. Fractions of peptides were contaminants), 554 and 695 proteins in total were identified collected by Proteineer fc II (Bruker Daltonik GmbH, from the WUS samples of volunteers with DMFT = 0 and Germany) which collected discrete fractions of the eluted DMFT > 0, respectively. The number of proteins found in peptides on MALDI target MTP AnchorChip 800/384 all samples within each group differed between them, i.e., TF (Bruker Daltonik GmbH, Germany) every 20 s in the a 179 for DMFT = 0 vs. 223 for DMFT > 0 identified proteins. 40–148 min interval. Subsequently, MALDI target was The details for the proteins in both experimental groups inserted to mass spectrometer for measuring. are depicted in Supplementary material I (see Table I A and Table I B). To comprehensively study the proteome profile 2.8 MALDI-TOF/TOF analysis changes that occurred in caries-susceptible individuals (compared to caries-free), the whole proteomic data were Mass spectrometry analysis was performed using a subjected to different functional analysis tools. MALDI-TOF/TOF UltrafleXtreme (Bruker Daltonik GmbH, Protein annotations were obtained primarily from Germany). Spectra were acquired in reflectron positive UniProt 7.0 including accession and entry name. The Gene ion mode in the m/z range 700–3500 Da. Alpha-cyano- ID Conversion Tool of DAVID Bioinformatics Resources 6.7, 4-hydroxycinnamic acid (HCCA) mixture was used as the NIAID/NIH [28] was used to convert UniProt entry names to matrix. The 800 μl of the HCCA matrix solution for nano- gene IDs. Cytoscape environment (Cytoscape 3.4.0 [29]) for HPLC fractions was prepared by mixing: 748 μl of TA95 integrated models of biomolecular interaction networks (95% ACN + 5% water solution of 0.1% TFA), 36 μl HCCA with DyNet [30], Biological Network saturated in TA90 (90% ACN + 10% water solution of (BiNGO) [31] and ReactomeFIPlugIn plug-ins were used 0.1% TFA), 8 μl of 10% water solution of TFA and 8 μl to describe different aspects of functional annotation of of 100 mM NH H PO dissolved in water. The 800 μl of 4 2 4 networks. HCCA matrix solution for calibrant spots was prepared In pair wise mode supporting the presence or absence using same procedure but TA85 solution (85% ACN + of nodes and edge attribute, the DyNet application 15% water solution of 0.1% TFA) was used instead of provides comparison of two network states [30]. That is TA95. External mass calibration was performed using the why the DyNet plug-in was used for the visualization and Peptide Calibration Standard II (Bruker Daltonik GmbH, for the highlighting differences between the two networks Germany). 252 G. Laputková, et al. of individuals with DMFT = 0 and DMFT > 0 based on with the aid of the ReactomeFIPlugIn tool for further node/edge presence attribute. The network encompassed analysis. GO analysis of biological processes was carried 208 functional partners (nodes) and 515 edges represented out with the aid of ReactomeFIPlugIn tool as well. in green (DMFT = 0), red (DMFT > 0) and grey color (both), depicted in Figure 1 A. In pair wise mode, DyNet is used to highlight the 3.1 Enrichment of the cell component cate- most variable nodes and edges on the network, using a gories assessed by GO color gradient of red (Figure 1 B). Dn-score supports the identification of the most rewired nodes (nodes with most Enrichment maps (Figure 2 A, B) were analyzed by varying edge connections) in a dynamic network or, more searching for the changes associated with the potential specifically, the most dynamic neighborhoods [30]. risk factors connected to dental caries. 87 and 83 GO GO term enrichment analysis of three categories, cell component terms were found enriched in the group i.e., biological process, molecular function and cellular with DMFT = 0 and DMFT > 0, respectively. 10 GO cell component for both groups of individuals using BiNGO component categories with the smallest p values were plug-in (version 2.44) in Cytoscape software environment selected as significant, depicted in the Table 1 and Table 2. was carried out. These annotation clusters facilitate GO terms in both groups were mainly related to the visualization of the connections associated with different extracellular region, extracellular space, and proteins in various categories within GO. The enrichment cytoskeleton. Membrane-bound vesicles and cytoplasmic analysis was done using a hypergeometric distribution membrane-bound vesicle proteins were included in the test. GO terms were selected after correcting for a multiple top ten with the smallest p-values of DMFT = 0, but not term testing with a Benjamini and a Hochberg FDR using DMFT > 0 group, while cytosol and cytoplasmic part GO Bonferroni correction at significance level of p<0.05. GO terms were found significantly enriched in the group with terms were visualized as networks that were sub-clustered DMFT > 0.

Figure 1. DyNet visualization of union gene/protein network of individuals with DMFT = 0 and DMFT > 0 (A). The red nodes/edges are present only in the case of DMFT > 0; green nodes/edges are present only in DMFT = 0; grey nodes/edges are present in both. DyNet highlights nodes which are most rewired (B) (more red ‒ nodes with higher varying edge connections) via the Dn-score. Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 253

Figure 2. Cell component networks (GO terms) for proteins in DMFT = 0 (A) and DMFT > 0 (B) group. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term (to distinguish first 10 component categories with smallest p-value apart from others, they were marked with red color). 254 G. Laputková, et al.

Table 1. GO cell component categories with the smallest p-values in the group of individuals with DMFT = 0.

GO-ID p-value Corr. p-value Description 5576 2.4110E-19 7.7392E-17 extracellular region 5856 3.5256E-14 5.6585E-12 cytoskeleton 5615 6.7538E-14 7.2265E-12 extracellular space 5737 1.7021E-13 1.3659E-11 cytoplasm 15629 4.3445E-13 2.7892E-11 actin cytoskeleton 44421 8.7725E-13 4.6933E-11 extracellular region part 44430 5.8347E-11 2.6756E-9 cytoskeletal part 48770 5.2515E-10 1.8731E-8 pigment granule 42470 5.2515E-10 1.8731E-8 melanosome 31988 8.3776E-10 2.6892E-8 membrane-bounded vesicle

Table 2. GO cell component categories with the smallest p-values in the group of individuals with DMFT > 0.

GO-ID p-value Corr. p-value Description

5576 6.2470E-18 2.0365E-15 extracellular region 5615 4.2004E-16 6.8467E-14 extracellular space 5737 1.7464E-15 1.8977E-13 cytoplasm 5856 1.5174E-14 1.2367E-12 cytoskeleton 15629 5.0041E-13 3.2627E-11 actin cytoskeleton 44421 6.7101E-13 3.6458E-11 extracellular region part 44430 6.7416E-12 2.9687E-10 cytoskeletal part 5829 7.2851E-12 2.9687E-10 cytosol 48770 2.8894E-11 9.4195E-10 pigment granule 42470 2.8894E-11 9.4195E-10 melanosome

3.2 Enrichment of the molecular function activity of proteolytic peptidases or its regulation. In contrast categories assessed by GO to WUS from caries-susceptible individuals, carbohydrate binding was included in the top ten functions of caries-free In order to investigate the specific molecular functions individuals. In the caries-susceptible group, binding, actin represented among the proteins identified in the saliva binding, antigen binding and calcium ion binding were of caries-free and caries-susceptible individuals, analysis included in the top ten with the smallest p-values. of molecular function annotation was performed. The According to Gao et al. [32] salivary proteins are 84 and the 70 GO molecular function terms were found responsible for approximately 50% of the calcium enriched in the group with DMFT = 0 and DMFT > 0, concentration of dental plaque. As such, a change in the respectively. Again, 10 GO molecular function categories protein profile could affect calcium-binding sites. Thus, with the smallest p-values were chosen as significant calcium-binding proteins from saliva can without a doubt (Table 3 and Table 4). As shown in Figures 3 and 4, serve as a template for mineral growth in dental biofilms, and Tables 3 and 4, protein binding, cytoskeletal requiring further assessment [33]. protein binding, endopeptidase activity and structural Also, searching GO terms for molecular functions constituent of cytoskeleton categories were ranked as the in the OralCard web information system (http:// most significant in both studied groups. bioinformatics.ua.pt/OralCard/diseases/view/68003731) The rest of the terms of caries-free saliva samples and corresponding proteins associated with dental caries predominantly contained molecular function related to the was carried out (Table 5). Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 255

Table 3. GO molecular function categories with the smallest p-values in the group of individuals with DMFT = 0.

GO-ID p-value Corr. p-value Description

5515 8.2728E-12 2.8833E-9 protein binding 8092 1.3046E-11 2.8833E-9 cytoskeletal protein binding 4175 5.4058E-11 7.9646E-9 endopeptidase activity 5200 1.9339E-9 2.1370E-7 structural constituent of cytoskeleton 30246 1.1458E-8 8.6628E-7 carbohydrate binding 70011 1.1759E-8 8.6628E-7 peptidase activity, acting on L- peptides 4866 1.4698E-8 8.7708E-7 endopeptidase inhibitor activity 61135 1.5875E-8 8.7708E-7 endopeptidase regulator activity 8233 2.1641E-8 1.0628E-6 peptidase activity 30414 2.8791E-8 1.2726E-6 peptidase inhibitor activity

Table 4. GO molecular function categories with the smallest p-values in the group of individuals with DMFT > 0.

GO-ID p-value Corr. p-value Description

5200 1.5055E-13 7.1964E-11 structural constituent of cytoskeleton 5515 8.8528E-13 2.1158E-10 protein binding 8092 1.4434E-9 2.2998E-7 cytoskeletal protein binding 4175 1.6659E-8 1.9907E-6 endopeptidase activity 4857 2.0374E-7 1.9477E-5 enzyme inhibitor activity 5509 3.4614E-7 2.7576E-5 calcium ion binding 3823 4.2710E-7 2.9165E-5 antigen binding 4866 1.2538E-6 5.9502E-5 endopeptidase inhibitor activity 5488 1.2562E-6 5.9502E-5 binding 61135 1.3414E-6 5.9502E-5 endopeptidase regulator activity

To further expand our knowledge about the network intramolecular transferase activity, phosphotransferases associated with caries, the networks were sub-clustered bisphosphoglycerate phosphatase activity, with the aid of the ReactomeFIPlugIn tool and the modules 2,3-bisphosphoglycerate-dependent phosphoglycerate were analyzed. In module 1 of the caries-susceptible mutase activity, intramolecular transferase activity) group, a branch of cytokine binding and gene were observed only in the oral environment of caries-free products not seen in the caries-free network was individuals. observed. Interleukin-11 binding, interleukin-11 receptor Water-insoluble glucans produced by cariogenic activity and interleukin-27 receptor activity responsible for species S. mutans play an important role in the formation the regulation of immune and inflammatory responses to of dental biofilm and adhesion of biofilm to tooth surfaces. infections were included into this sub-cluster in contrast Hydrolase-active glucanohydrolases (α-1,3-glucanase, to caries free-individuals. α-1,6-glucanase) are potentially useful for dental caries In module 2, there were also observed changes that could prevention. Bi-functional chimeric glucanase, composed relate to the mechanism of caries production. Some genes of α-1,3-glucanase and α-1,6-glucanase, can reduce the and gene products annotated to hydrolase activity (i.e., formation of the total amount of water-insoluble glucan phosphoric ester hydrolase activity, bisphosphoglycerate in a dose-dependent manner, and more effectively phosphatase activity, 2,3-bisphospho-D-glycerate decompose biofilm than a mixture of α-1,3-glucanase and 2-phosphohydrolase activity) and intramolecule α-1,6-glucanase [34]. transferase activity (i.e., activity, Transferases belong to that metabolize 256 G. Laputková, et al.

Figure 3. Molecular function networks (GO terms) for proteins of caries-free individuals. Color relates to the p-value for the statistical signifi- cance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term (to distinguish first 10 component categories with smallest p-value apart from others, they were marked with red color). sucrose into water insoluble and soluble glucans, which 3.3 Enrichment of biological process catego- are an integral measure of the biofilm formation initiated ries assessed by GO by S. mutans. Looking for an ideal treatment preventing dental caries, efforts were taken to explore therapeutic The hierarchical networks depicted in the Figure 5 and approaches that selectively inhibit the biofilm formation Figure 6 show the variety and interdependence of the process while preserving the natural bacterial flora of biological processes in terms of GO. The 507 and the the mouth. Confirming the importance of the presence 397 GO biological process terms were found enriched of transferases in the process of forming tooth decay, it in the group with DMFT = 0 and DMFT > 0, respectively. was found that S. mutans glucosyl transferases activity The 20 GO biological process categories with the smallest can be inhibited by a group of hydroxychalcones. Leaving p-values were selected as significant (Table 6 and Table commensal and/or beneficial microbes intact, they can 7). As shown in the Table 6 and Table 7, in the first group serve as potential anti-caries agents [35]. these processes associated with the of Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 257

Figure 4. Molecular function networks (GO terms) for proteins of caries-susceptible individuals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term (to distinguish first 10 component categories with smallest p-value apart from others, they were marked with red color). carbohydrates dominate. The following processes are not The sub-clusters of networks were selected for detailed even included in the top 20 for the caries-experienced analysis. The examination of module 0 also confirmed group: catabolic, monosaccharide metabolic, differences in some branches of the network related to the cellular carbohydrate catabolic, hexose catabolic, metabolic processes and their regulation, as well as the monosaccharide catabolic, and . regulation of catabolic processes. Figure 7 A, B shows the Nevertheless, response to chemical stimulus, response functional sub-clusters of module 0 representing altered to stimulus, immune system process, immune response, metabolic processes. Table 8 displays a list of GO terms defense response, inflammatory response, immune of those processes that occurred in caries-free individuals, effector process and regulation of immune system process but were not recorded in group of caries-susceptible were ranked as the most significant per the results in the people. However, by searching GO terms for biological group of caries-experienced individuals. processes in the OralCard web information system, only 258 G. Laputková, et al.

Table 5. GO terms for molecular functions associated with dental caries found in OralCard.

Molecular functions DMFT = 0 DMFT > 0 protein binding P11021 ‒ 78 kDa glucose-regulated protein, P11021 ‒ 78 kDa glucose-regulated protein, P01040 ‒ cystatin-A, P01040 ‒ cystatin-A, P04083 ‒ P04083 ‒ annexin A1 endopeptidase activity O14773 ‒ tripeptidyl-peptidase 1 O14773 ‒ tripeptidyl-peptidase 1 endopeptidase inhibitor activity - P01024 ‒ complement C3 constituent of cytoskeleton P02533 ‒ keratin, type I cytoskeletal 14, - P08779 ‒ keratin, type I cytoskeletal 16, P35908 ‒ keratin, type II cytoskeletal 2 epidermal, P60709 ‒ actin, cytoplasmic 1 peptidase activity P01024 ‒ complement C3 - endopeptidase inhibitor activity structural constituent of - P02533 ‒ keratin, type I cytoskeletal 14, cytoskeleton P08779 ‒ keratin, type I cytoskeletal 16, P35908 ‒ keratin, type II cytoskeletal 2 epidermal, P60709 ‒ actin, cytoplasmic 1 calcium ion binding - P11021 ‒ 78 kDa glucose-regulated protein, P04083 ‒ annexin A1, Q14515 ‒ SPARC-like protein 1, P07996 ‒ thrombospondin-1, Q08188 ‒ protein- glutamine gamma-glutamyltransferase E antigen binding - P01625 ‒ Ig kappa chain V-IV region Len, P01591 ‒ immunoglobulin J chain, P01593 ‒ Ig kappa chain V-I region AG, P01596 ‒ Ig kappa chain V-I region CAR, P01614 ‒ Ig kappa chain V-II region Cum, P01620 ‒ Ig kappa chain V-III region SIE, P01703 ‒ Ig lambda chain V-I region NEWM, P01766 ‒ Ig heavy chain V-III region BRO, P01876 ‒ Ig alpha-1 chain C region, P01877 ‒ Ig alpha-2 chain C region, P04208 ‒ Ig lambda chain V-I region WAH, P25311 ‒ zinc-alpha-2-glycoprotein, P80748 ‒ Ig lambda chain V-III region LOI

Table 6. GO biological process categories with the smallest p-values in the group of individuals with DMFT = 0. (* biological processes not included in top 20 DMFT > 0 group).

GO-ID p-value Corr. p-value Description

65008 3.31E-14 6.911E-11 regulation of biological quality 6006 1.36E-13 1.1252E-10 glucose metabolic process 2376 1.73E-13 1.1252E-10 immune system process 16052 2.16E-13 1.1252E-10 carbohydrate catabolic process 42221 4.21E-13 1.759E-10 response to chemical stimulus 19318 5.76E-13 2.0038E-10 hexose metabolic process 9611 1.12E-12 3.3448E-10 response to wounding 50896 1.99E-12 5.1955E-10 response to stimulus 6007 4.93E-12 1.1421E-09 glucose catabolic process* 5996 7.51E-12 1.5675E-09 monosaccharide metabolic process* 6928 1.48E-11 2.8079E-09 cellular component movement* 48518 1.64E-11 2.8486E-09 positive regulation of biological process 44275 2.95E-11 4.7389E-09 cellular carbohydrate catabolic process* 19320 3.65E-11 5.4336E-09 hexose catabolic process* 46365 5.21E-11 7.2432E-09 monosaccharide catabolic process* 6954 7.55E-11 9.8521E-09 inflammatory response 6952 1.63E-10 1.9982E-08 defense response 30036 2.98E-10 3.4503E-08 actin cytoskeleton organization* 6096 3.37E-10 3.4764E-08 glycolysis* 6950 3.39E-10 3.4764E-08 response to stress Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 259

Figure 5. Biological processes networks (GO terms) for proteins in caries-free individuals. Color relates to the p-value for the statistical signi- ficance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term.

Figure 6. Biological processes networks (GO terms) for proteins caries-susceptible individuals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term. 260 G. Laputková, et al.

Table 7. GO biological process categories with the smallest p-values in the group of individuals with DMFT > 0. (*biological processes not included in top 20 DMFT = 0 group).

GO-ID p-value Corr. p-value Description

65008 4.22E-14 8.42E-11 regulation of biological quality 42221 7.98E-14 8.42E-11 response to chemical stimulus 50896 1.13E-13 8.42E-11 response to stimulus 2376 1.64E-13 9.16E-11 immune system process 9611 3.17E-13 1.42E-10 response to wounding 6955 1.22E-11 4.55E-09 immune response* 6950 1.50E-11 4.79E-09 response to stress 48518 1.42E-10 3.96E-08 positive regulation of biological process 10033 2.87E-10 6.52E-08 response to organic substance* 6952 2.91E-10 6.52E-08 defense response 6928 3.62E-10 7.36E-08 cellular component movement* 6954 6.39E-10 1.19E-07 inflammatory response 51128 5.27E-09 8.58E-07 regulation of cellular component organization* 6006 5.36E-09 8.58E-07 glucose metabolic process 16052 1.05E-08 1.47E-06 carbohydrate catabolic process 2252 1.05E-08 1.47E-06 immune effector process* 34637 1.22E-08 1.61E-06 cellular carbohydrate biosynthetic process* 19318 1.50E-08 1.87E-06 hexose metabolic process 2682 2.51E-08 2.96E-06 regulation of immune system process* 51246 3.85E-08 4.31E-06 regulation of protein metabolic process*

Figure 7. Biological processes (GO terms) of part of sub-networks of module 0 for proteins of caries-free (A) and caries-susceptible (B) indivi- duals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term. Nodes in white are not significantly overrepresented; they are incorporated to show other nodes in the framework of GO hierarchy. Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 261

Table 8. GO biological process categories of sub-networks of module 0 for proteins of caries-free individuals with DMFT = 0.

GO-ID p-value Corr. p-value Description Genes in test set

16052 9.34E-04 1.36E-02 carbohydrate catabolic process HK3|G6PD|PFKL 44275 4.14E-04 7.64E-03 cellular carbohydrate catabolic process HK3|G6PD|PFKL 44262 4.61E-03 3.08E-02 cellular carbohydrate metabolic process PPP1CB|HK3|G6PD|PFKL 6014 7.25E-03 4.10E-02 D-ribose metabolic process G6PD 6007 1.18E-04 4.58E-03 glucose catabolic process HK3|G6PD|PFKL 6006 1.31E-04 4.83E-03 glucose metabolic process PPP1CB|HK3|G6PD|PFKL 6096 2.62E-03 2.48E-02 glycolysis HK3|PFKL 19318 3.24E-04 7.52E-03 hexose metabolic process PPP1CB|HK3|G6PD|PFKL 2349 1.82E-03 1.87E-02 histamine production involved in inflammatory response YWHAZ inflammatory response 32501 1.07E-03 1.42E-02 multicellular organismal process YWHAE|G6PD|CALML5|PEBP1|SORBS1|Y WHAZ|GABARAP|MYL12B|EHD1|HK3|AL MS1|MYH9|SFN|CALM1|CALM2|CNGB1 6936 1.65E-04 5.50E-03 muscle contraction SORBS1|CALM1|CALM2|MYL12B 90004 9.06E-03 4.74E-02 positive regulation of establishment of protein localization in SORBS1 plasma membrane 32414 2.07E-04 6.32E-03 positive regulation of ion transmembrane transporter activity CALM1|CALM2 10962 3.75E-04 7.52E-03 regulation of glucan biosynthetic process PPP1CB|SORBS1 10906 2.04E-03 2.01E-02 regulation of glucose metabolic process PPP1CB|SORBS1 5979 3.75E-04 7.52E-03 regulation of glycogen biosynthetic process PPP1CB|SORBS1 5981 9.06E-03 4.74E-02 regulation of glycogen catabolic process PPP1CB 34765 1.93E-03 1.93E-02 regulation of ion transmembrane transport CALM1|CALM2 32412 1.52E-03 1.87E-02 regulation of ion transmembrane transporter activity CALM1|CALM2 32885 3.75E-04 7.52E-03 regulation of polysaccharide biosynthetic process PPP1CB|SORBS1 32881 4.25E-04 7.64E-03 regulation of polysaccharide metabolic process PPP1CB|SORBS1 51279 4.25E-04 7.64E-03 regulation of release of sequestered calcium ion into cytosol CALM1|CALM2 34762 2.38E-03 2.28E-02 regulation of transmembrane transport CALM1|CALM2 22898 1.72E-03 1.87E-02 regulation of transmembrane transporter activity CALM1|CALM2

the term glycolysis (P04406 ‒ glyceraldehyde-3-phosphate cytokines, some of which promote cell-mediated immune dehydrogenase) was found as being related to dental functions, organism response to microbial infection, or caries. can induce a class of protein-degrading enzymes known The influence of other proteins involved in metabolic as matrix metalloproteinases [36]. processes and in the regulation of metabolic and catabolic The significantly elevated concentration of the pro- processes of carbohydrates of caries-free individuals inflammatory cytokines interleukin-6, interleukin-8, and (Table 8) for caries protection was also included. tumor necrosis factor α found in WUS of caries-susceptible Module 1 of GO terms of caries-susceptible individuals subjects, compared to caries-free individuals, confirms contained such biological processes as interferon-gamma their potential impact on the formation of dental caries biosynthetic process, interferon-gamma production, [37]. interleukin-13 biosynthetic process, interleukin-13 Also, the up-regulation of inflammatory cytokines production, interleukin-2 biosynthetic process, and that carry the converged inflammatory signal in the interleukin-27-mediated signaling pathway, which were odontoblast layer of human teeth suffering with caries not recorded in the caries-free group. Regarding these was observed [38]. In another study [39], it was found biological processes, all are linked to the production of that cytokines increase defensive capacity, including 262 G. Laputková, et al. antimicrobial peptide production, to protect the tooth. in the saliva of the caries-susceptible group. Generally, Controversially, dental caries was not correlated with found in are cationic antimicrobial salivary or serum concentrations of the studied cytokines peptides, but their role in the protection of dental enamel of children aged 6-12 years. is still unclear. Similarly unknown is the role of azurocidin identified in both of our experimental groups. Azurocidin is a 3.4 Assessment of proteins using GeneAna- neutrophil granule-derived antibacterial and monocyte- lysis tool and OralCard and fibroblast-specific chemotactic glycoprotein. There could be a relationship between azurocidin incidence GeneAnalytics tools (https://ga.genecards.org/#) that and dental-caries occurrence as it has been detected provide information about genes of interest as well as among unique proteins in caries-free children [40]. precise analysis of how these genes are connected to Several members of the S100 multigenic calcium- different diseases and OralCard web information system modulated protein family were also detected, as: S100- (http://bioinformatics.ua.pt/OralCard/diseases/view A2, A7, A8, A9, A11 and A12 (in the caries-free group) /68003731) were further used to study our experimental and S100-A4, A6, A7, A8, A9, A11 and A12 (in the caries- data. susceptible group). The family consists of Searching GeneAnalytics database revealed 7 proteins 24 members functionally distributed into those which encompassed in both groups that are directly associated only exert intracellular regulatory effects, those with with dental caries or processes related to them: cystatin intracellular and extracellular functions and those SN, lactoperoxidase, lactotransferrin, lysozyme and which mainly exert extracellular regulatory effects mucin 7, secreted, are related to dental caries; [41]. Their possible relationship to dental-caries was and glucose-6-phosphate isomerase are related to dentine confirmed by immuno-histochemical studies in carious erosion; glucose-6-phosphate isomerase is related to pulpal tissue [42]. PCR analysis in carious and healthy enamel erosion; glucose-6-phosphate isomerase is pulpal tissue samples of the S100 family members related to root caries; and glucose-6-phosphate isomerase S100-A8, S100-A9, S100A-12, and S100-A13, indicated is related to tooth erosion. that genes tested were more abundantly expressed in OralCard database (67 stated proteins) was also used carious teeth. The authors showed by gene expression to assess our data and compared well with them (see Table analyses in immune system cells that S100-A8 and II A in Supplementary material II). The table of caries- the S100-A8/S100-A9 complex were mainly expressed experienced individuals included Ig kappa chain V-II by infiltrating neutrophils and revealed that bacterial region Cum, Ig lambda chain V-I region NEWM, Ig lambda activation of neutrophils caused up-regulation of S100- chain V-III region LOI, and keratin, type I cytoskeletal 17, A8, S100-A9, and S100-A13. that were not found in the group with DMSF=0, while Profilin-1 which is classified to the group of proteins histone H4 and chitinase-3-like protein 2 were observed responsible for focal adhesion appears also to be of only in the group with DMSF>0. interest from the point of caries development [42]. In A number of identified salivary proteins that are not another study aimed to compare the proteins of caries-free listed in GeneAnalytics or OralCard are involved in host with caries-susceptible saliva, profilin-1 was identified as defense control in the oral cavity and prevent the growth a unique protein of caries-free samples [18]. Conversely, of microorganisms. Therefore, they could play a role in we identified profilin-1 in our both experimental groups. protecting tooth structure from caries by providing a PRPs are divided into the three classes: acidic, natural antibiotic barrier. They may also have a function basic, and basic glycosylated, representing the most to keep overall bacteria within reasonable limits and help heterogeneous family of human salivary proteins [43]. prevent biofilm formation. From our results, the incidence Whatever the role of PRP species is, it should be noted of neutrophil defensin-1 and neutrophil defensin-4 that PRPs are the most conserved oral salivary proteins in caries-free subjects is consistent with the study of among mammals. However, further investigations are Hong et al. [18]. They recently confirmed the presence needed to clarify their different functions in the oral cavity of neutrophil defensin-1 in saliva samples of caries- [44]. The participation of acidic PRPs in the formation of free subjects but not of caries-positive subjects while acquired enamel and the development of dental erosion, studying qualitatively differential profiles of salivary functioning as predominant pellicle precursor proteins, proteins with affinity to S. mutans lipoteichoic acid [18]. has been well established. They are involved in the Controversially, we identified neutrophil defensin-1 also development of the basal layer of the acquired pellicle Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment 263 and control dental erosion by modulating calcium were observed only in the oral environment of caries- and phosphate concentration within the oral cavity free individuals. In contrast to WUS from the caries- [45]. Moreover, according to Zakhary et al. [46], there free group, the caries-susceptible samples network is a correlation between acidic PRP alleles of the PRH1 contained a branch of cytokine binding genes and gene (Db) from parotid saliva and caries susceptibility: products responsible for the regulation of immune and Db-negative individuals had significantly more caries inflammatory responses to infections, which were not prevalence. observed in the caries-free network. In our experiment, the proline-rich protein 1, proline- GO analysis of biological processes further rich protein 4 and three small PRP 2B, 2D and 3 were revealed differences in some branches of the network identified in caries-free subjects while PRP 4 and small corresponding to metabolic processes and the regulation PRP 2A, 2B, 2D, 2E, 2F and 3 were observed in caries- of metabolic and catabolic processes of carbohydrates susceptible subjects. in the caries-free group. In contrast, such biological processes as immune system process, immune response, defense response, inflammatory response, immune 4 Conclusions effector process and regulation of immune system process were ranked as the most significant per the results in the Only a few research efforts have been studying the protein group of individuals with caries. contents of the oral cavity and/or saliva for comparing Therefore, this study contributes toward the current differences between groups of caries-free and caries- understanding of tooth protection mechanisms and susceptible individuals to date. Also, a significant finding additionally provides information about possible risk on a particular protein or group of proteins supposedly factors associated with the development of dental caries. responsible for the resistance or vice versa for the Indeed, further studies in comparative proteomics of potential risks leading to dental caries is still very limited. human teeth in connection to dental caries would be Moreover, the studies in this field are often controversial. necessary and is currently under investigation in our lab. Dental caries is a disease with multi-factorial, thus, complex etiology associated with the effects of multiple Conflict of interest: All authors declare no conflict of genes in combination with behavioral factors and oral interest. hygiene habits. Therefore, we studied different aspects of functional Sources of funding: This work was supported by annotation of biomolecular interaction networks. the Agency of the Slovak Ministry of Education for GO term enrichment analysis of three categories, i.e., the Structural Funds of the EU [project number ITMS biological process, molecular function and cellular 26220220143 (100%)]. component for both caries-free and caries-susceptible groups were carried out. These annotation clusters Acknowledgements: The authors gratefully facilitate visualization of the connections associated acknowledge the volunteers – students of Dental with different proteins in various categories within GO. A Medicine of Medical Faculty, P. J. Šafárik University in comparative analysis to assess the potential risk factors Košice − for participating in this research. connected to dental caries of the WUS samples of caries- free and caries-susceptible volunteers of older adolescent age with permanent dentition revealed qualitative Abbreviations differences between these two proteomes. Although the number and type of proteins identified were different, the ACN − Acetonitrile annotation per GO term revealed some biological paths DMFT − Decay-missing-filled teeth index and molecular functions being affected for patients who DTT − DL-Dithiothreitol experienced dental caries. FA − Formic acid Inspecting molecular functions of caries-free saliva GO − Gene ontology samples revealed that the top 10 GO molecular function HCCA − Alpha-cyano-4-hydroxycinnamic acid categories, chosen as significant, predominantly PRP − Proline-rich proteins contained terms related to the activity of proteolytic SPE − Solid phase extraction peptidases or regulation of their activity. Some genes TFA − Trifluoroacetic acid and gene products annotated to hydrolase activity WUS − Whole unstimulated saliva 264 G. Laputková, et al.

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