A variety of bacterial and mammalian derived proteases are present in the human mouth. These proteolytic enzymes influence the structure and activity of several salivary and pellicle components. Many of these proteolytic enzymes are thiol proteases. Salivary secretions contain a family of cysteine containing phosphoproteins, some of which at least are members of the """"""""cystatin"""""""" family of thiol protease inhibitors. These proteins, which ave been shown to be pellicle proteases; and hence serve to help maintain the integrity of the protective roles of saliva and pellicle. The immediate specific aims of this work are: (1) to determine whether the phosphorylation state of the major salivary cystatins influences their ability to function as thiol protease inhibitors or to interact with metal ions and hydroxyapatite, and (2) to establish whether the thiol protease inhibitory activity of hydroxyapatite-bound cystatins is altered compared to that exhibited in solution. The overall experimental design is to isolate the 3 major classes of salivary cystatins using gel filtration and ion exchange chromatography, and to purify the major protein component from each class using reversed phase HPLC. For each major component isolated, the number covalently bound phosphate groups per molecule will be determined. The phosphate content will be correlated with the thiol protease inhibitory activity, determined in solution using a fluorescent thiol protease assay, and the ability of the component to bind metal ions, determined using fluorescence titrations and competition binding studies. The binding of each component to hydroxyapatite will also be correlated to the phosphorylation state. The thiol protease inhibitory activity of the hydroxyapatite-bound cystatins will be determined. The achievement of these aims will indicate whether salivary cystatins may play a thiol protease inhibitory role in vivo and will provide the material and information necessary for the anticipated long term goals of the project. These goals are to explore the role phosphorylation may play in the synthesis, function, and in vivo localization of the various salivary cystatins.
