Abstract
With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein–protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein–protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance.
Highlights
The LC-ESI-MS/MS analysis of those potential histatin 5 protein partners obtained by both Co-IP and pull-down techniques resulted in the identification of 83 different proteins that came from the “in-solution” and “in-gel” experiments
One-third of the identified histatin 5 protein partners exhibited acidic characteristics ranging in pI between 5.0 to 6.7 (Fig 1)
Histatin 1 is generated from HIS 1 gene while histatin 5 is a degradation product from histatin 3 that is generated from the HIS2 gene [33]
Summary
Recent efforts in salivary research have resulted in the elucidation and characterization of the proteomes of the major gland human salivary secretions and whole saliva [1,2,3,4,5,6] by classicalPLOS ONE | DOI:10.1371/journal.pone.0142517 November 6, 2015Histatin 5 Interactome biochemical methods [7,8,9,10] as well as more advanced approaches [1,5,6,11,12,13,14]. Understanding the consequences of protein interactions is a crucial role for the development of novel therapeutics approaches [20]. A previous study mapped protein interactions for 338 human bait proteins that were selected based on known disease and functional associations. Other studies used the same approach to map protein-protein interactions in yeast, creating unique data sets for biology and extrapolation into mammalian biology [22,23]. Salivary protein complexes are described [15,16,17] and a suitable biological function for these complexes is to serve as a mechanism between salivary protein partners for protection from oral proteolysis; and/or to play a role in the delivery of salivary proteins to different locations in the oral cavity
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