Global proteome comparative analysis of periodontal tissues from deciduous and permanent human teeth

Author(s)

Priscila Alves Giovani

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2020-12-14

Examining board members

Kamila Rosamilia Kantovitz; Saul Martins de Paiva; Isabela Almeida Pordeus; Eduardo César Almada Santos; Carolina Steiner Oliveira

• Advisor: Kamila Rosamilia Kantovitz; Regina Maria Puppin Rontani

Abstract

The periodontal ligament (PDL) is a highly specialized connective tissue that connects the tooth to the alveolar bone, while the dental cementum (DC) is a mineralized, avascular connective tissue that covers the root dentin and its main function is the insertion of fibers from the periodontal ligament to the tooth root. Together, these tissues play a critical role in tooth anchorage. There is a lack of information regarding the molecular signature of deciduous (DecPDL) and permanent (PermPDL) periodontal tissues. The present thesis characterized the proteome of the cell membrane of the PDL and the extracellular matrix of the DC in order to identify distinctions between the dentitions. Proteins were extracted from the cell membrane of primary cultures for DecPDL (n=3) and PermPDL (n=3) and from extracellular matrix of dental cementum 25 Dec and 12 Perm, grouped in pools for DecDC (n=5) and PermDC (n=4) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The beta-binomial test applied to spectrum counts determined differentially expressed proteins (PDE) (p <0,05). PDL proteome findings were validated by quantitative polymerase chain reaction and Western blot assays in fresh human tissues (n=8) and primary cell cultures (n=6). In addition, confocal microscopy was used to verify the expression of target factors in the PDL cell cultures. Otherwise, Immunohistochemistry was applied to validate selected proteins of DC. In PDL a total of 752 proteins were detected: 142 exclusive to DecPDL, 115 exclusive to PermPDL and 495 common. 23 were PDE: three with higher abundance in DecPDL and 10 found exclusively in DecPDL cells; two proteins with higher abundance in PermPDL and eight found to be exclusive for PermPDL cells. Comparative gene ontology enrichment analysis (GO) in PDL evidenced that the most stickling differences involved "endomembrane system" (PICALM, STX4, and LRP10), "hydrolase activity" (NCSTN and XRCC6), "protein binding" (PICALM, STX4, GPNMB, VASP, ESYT2, and LRRC15), and "isomerase activity" (FKBP8). At the transcript level, high PICALM in DecPDL and ESYT2 and LRRC15 in PermPDL, reproducing the proteomic findings in fresh PDL tissues. Furthermore, Western blot analysis confirmed increased levels of PICALM, LRRC15, and ESYT2 in cells and/or fresh tissues, and confocal microscopy confirmed the trends for PICALM and LRRC15 expression in PDL cells. In DC a total of 510 proteins were identified: 123 exclusive to DecDC, 128 exclusive to PermDC and 259 common. Among the 60 PDE, 17 were higher in DecDC, including MPO (found exclusively in DecDC [p < 0.05]), whereas 43 were higher in PermDC, including DCN and BGLAP (higher in PermDC [p < 0.05]). Overall, GO analysis indicated that proteins were related to Biological Processes that included localization and response to stress; and PDE were enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected PDE in human teeth. We report the first comprehensive characterization of the membrane protein machinery of DecPDL vs. PermPDL cells, and of extracellular matrix of DecDC vs. PermDC. Together, we identified a distinct molecular profile for these dentitions, including unique proteins (AU)

• FAPESP's process: 16/02942-1 - Global Proteome Profile of Periodontal Ligament.
• Grantee: Priscila Alves Giovani
• Support Opportunities: Scholarships in Brazil - Doctorate