In Vitro Identification of Histatin 5 Salivary Complexes

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.

Fig 1. Percentage of histatin 5 protein partners according to Isoelectric Point (pI).

The histatin 5 protein partners were divided according to the pI ranging from 5.0 to 6.7, 6.8 to 7.2 or above 7.2.

Fig 2. C. albicans killing Assay.

C. albicans killing activity of histatin 5, amylase, histatin5/amylase complex prior incubate with C. albicans and histatin 5/amylase incubation with C. albicans. X-axis represents the concentration of histatin 5 complexed (or not). The complex was made based on 1:1 number of molecules from histatin 5 and amylase. Bars represent standard deviation of the mean, calculated from three independent experiments.

Fig 3. Hydrolysis of Starch.

Hydrolysis of starch by histatin 5, amylase and histatin 5/amylase complex. Different lower case letters denote statistical difference according to Tukey’s test. Bars represent standard deviation of the mean, calculated from three independent experiments.