Influence of periodontitis on salivary peptidome and proteases

• Autor(a) principal: Trindade, Fábio Jorge Sousa
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/13828
• topic: Bioquímica clínica; Periodontite; Péptidos; Saliva; Marcadores bioquímicos; Bactérias; Enzimas proteolíticas

Periodontitis is a complex immuno-inflammatory disease that results from a pre-established infection of gingiva, mainly due to Gram negative bacteria, which colonize deeper in gingival sulcus and latter in periodontal pocket. The progressive unresolved inflammation promote connective tissue loss and alveolar bone resorption, leading to several histopathological changes, namely destruction of periodontal ligament, deepening of periodontal pocket, bone loss and even tooth loss. Despite the efforts of the omics, until now there is no available biomarker for periodontitis, forcing diagnosis to continue based on certain clinical parameters such as clinical attachment level, probing depth, bleeding on probing and alveolar bone radiography. Peptidomic approaches seem promising to find surrogate markers for periodontitis. In that sense, saliva has been attracting researchers due to its diagnostic potential, ease and non-invasive nature of collection. The salivary peptidome is highly influenced by proteolytic events. In order to disclose the proteolytic events taking place in saliva, salivary peptidome was characterized and the salivary proteases were predicted applying, for the first time, filter-aided sample preparation (FASP) approach to saliva. Thus, saliva samples were incubated in spin filters for 18 or 115 hours, at 37 ºC, to promote saliva autolysis and the generation of novel peptides. In ex vivo conditions, proline-rich proteins (PRP), P-B peptide, histatin 1 and statherin came out as the most susceptible proteins to proteolysis. Peptide fragments were mainly attributed to cathepsins L1, K and MEP1A. The described endoProteoFASP approach avoids the use of synthetic peptides and exogenous proteases and could be very helpful in future studies targeting the characterization of salivary proteases and peptidome from pathophysiological conditions associated with remarkable proteolytic events. Following an endoProteoFASP approach and making also use of zymographic studies, the salivary peptidome and the proteolytic activity was studied in chronic periodontitis (CP). Overall, CP is associated with increased gelatinolytic and collagenolytic activity, which is mainly attributed to metalloproteases, remarkably MMP9. Proteomic and peptidomic data corroborated the inflammatory status, and demonstrated that intact histatin 1 may play an important role in the defense response against oral pathogens. The application of the endoProteoFASP approach to study the salivary peptidome of CP subjects resulted in the identification of 8 surrogate peptide markers, which may be used in multiplex to identify CP. These peptides belong to acidic PRP and to P-B peptide. Particularly, P-B peptide fragments exhibited domains with potential predicted antimicrobial activity, proposing a novel function to this protein. Therefore, the endoProteoFASP strategy looks promising for large-scale application to the study of the salivary degradome in CP.