Salivary proteomic profile and histatins 1 and 5 degradation in individuals with Down syndrome and periodontal disease

This study aimed to evaluate the proteomic profile and histatins 1 and 5 proteolysis in stimulated whole saliva of individuals with Down syndrome (DS) and non-syndromics (NS) in the presence and absence of periodontal disease (PD). Twenty-four individuals were selected and divided in the following groups (n=6): DS with PD (DSwPD), DS without PD (DSwtPD), NS with PD (NSwPD) and NS without PD (NSwtPD – control). First, periodontal condition and DMFT index were evaluated and 3.0 mL of stimulated whole saliva was collected. Then, part of whole saliva was used to microbiological analysis and the remaining samples were centrifuged in order to obtain the whole saliva supernatant (WSS), aliquoted and stored at -80ºC to proteomic and degradation assays. Levels of Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) were quantified by real-time polymerase chain reaction (qPCR). Spectrophotometric analyses were carried out with saliva pools of each group by using nLC-ESI-MS/MS (Liquid chromatography electrospray ionization tandem mass spectrometry). Protein degradation assay was carried out with synthetic histatins 1 or 5 added to WSS (1:10) followed by samples incubation at 37°C for 0, 0.5, 1.5, 4, 6, 8, 24 and 48 hours. Next, polyacrylamide cationic gel electrophoresis (PAGE) analysis and measurement of bands density (%) were performed. Kruskal-Wallis test complemented by Dunn's test was applied to analyze clinical and microbiological data. Total protein and histatins degradation were evaluated by using Anova followed by Tukey’s and Bonferroni post hoc tests, respectively (a=0.05). The acquired spectra obtained from proteomic analysis were searched against specific protein database and descriptive statistics was used. Individuals with DS showed reduced salivary flow rate (p=0.001). There was no significant differences for the DMFT index between groups (p=0.158), while it was noticed significant differences in periodontal parameters between groups with PD and without PD (p≤0.003). Regarding amounts of periodontopathogenic bacteria, there were no significant differences between groups to Aa (p=0.803). On the other hand, higher significant levels of Pg were verified in NSwPD group compared to groups without PD (p=0.022). Total protein concentration was significantly higher in DSwPD group (p≤0.0001). A total of 1855 proteins were identified in the pool of saliva samples, being 31 in common to all groups, however with different abundances. It was also verified 11 exclusive proteins in individuals with DS and 33 exclusive proteins in individuals with PD. It was noticed that the degradation occurs faster in the presence of PD for both histatins (p≤0.0001). Bands density were influenced by incubation period (p≤0.0001) and by the histatin type (p≤0.0001). A higher degradation rate of histatin 5 was observed in comparison to histatin 1, and as longer as incubation period the greater was the degradation rate of these histatins. In addition, it was observed a higher degradation rate for histatin 1 in the presence of DS (p=0.036). It can be concluded that: (1) there are differences in the abundance of some proteins in the presence of DS and PD, (2) histatin1 is more resistant to proteolysis than histatin 5, and (3) histatins 1 and 5 degradation occurs faster in the presence of PD and only histatin 1 degradation is influenced by the presence of DS. (AU)