Secretion of Salivary Proteins and their Interactions
Abstract
Saliva’s clinical application for disease diagnosis and monitoring is limited by incomplete knowledge of salivary protein interactions, the effect of stimulation on the salivary proteome, and if these factors impact protein identification. This thesis expands knowledge of the salivary interactome and effects of stimulation intensity and duration on parotid saliva’s proteome. First, previous in vitro studies identified 43 proteins in the histatin 1-protein network and demonstrated histatin 1’s increased stability in whole saliva when interacting with amylase. We hypothesized that protein-protein-interaction databases could enlarge the histatin 1-protein network. A comprehensive histatin 1-protein network was created using STRING database, merging previous in vitro complex partners with in-silico interactors. Thirty-seven novel histatin 1 interactors were identified, demonstrating STRING’s utility for studying protein-protein networks. Second, heterotypic complexes between amylase and MUC 5B, MUC 7, histatin 1 and histatin 5 have been described. Given amylase’s biochemical characteristics and abundance in saliva, we theorized that amylase interacted with other proteins. Affinity chromatography, gel electrophoresis, tryptic in-gel and in-solution digestion, and mass spectrometry were used. Sixty-six proteins were identified in whole saliva’s amylase-protein network. Acidic, low molecular weight proteins involved in host protection had preference in amylase’s complex formation. An inclusive amylase-protein network was constructed using STRING database, opening avenue for further studies about the amylase interactome. Third, stimulation intensity and duration affect the composition of salivary gland secretions. We questioned if the proteome of the parotid gland’s secretion was also affected by stimulation intensity and duration. Continuous parotid saliva secretion (0.25 and 1.00 ml/min) for 30 consecutive minutes was achieved. After in-solution digestion and mass spectrometry, five time points were used for proteome identification. Combining both flows, 169 proteins were identified. Stimulation intensity strongly affected 119 proteins, 44 were affected by both factors, and 4 by neither, suggesting possible protein-specific secretory mechanisms. This thesis demonstrates that salivary proteins participate in large complexes, that can be represented and expanded with aid of protein-protein-interaction databases. It also provides insights into the complexity of factors affecting saliva composition, such as stimulation, and highlights the importance of developing standardized protocols for salivary biomarker research.