Kinetics of Histatin Degradation in Human Saliva

Introduction:Histatins are human salivary antifungal proteins that are prone to proteolytic degradation upon their release into the oral cavity. Functional domains within the histatin structure have been elucidated, and evidence for their degradation in oral fluid has been reported. Objectives: To investigate the overall kinetic parameters for the degradation of histatins 1, 3 and 5 by whole saliva proteases and to structurally characterize the degradation products over time. Methods: Various concentrations of synthetic histatins were incubated with diluted pooled human whole saliva supernatant for various time intervals, and the resultant digests were analyzed by RP-HPLC and by LC-ESI-MS/MS. Results: Plotting of the initial histatin 1, 3 and 5 concentrations versus the degradation rates (V_i) yielded the following overal kinetic parameters: K_m values of 47 mmole.L^-1, 12 mmole.L^-1, and 5 mmole.L^-1; V_max values of 15 nmole.L^-1.s^-1, 21 nmole.L^-1.s^-1, and 17 nmole.L^-1.s^-1; and catalytic efficiencies (V_max/K_m) of 0.32x10^-3.s^-1, 1.8x10^-3.s^-1, and 3.4x10^-3.s^-1, respectively. The amino acid sequences of the proteolytic fragments of histatin 1, 3 and 5 determined by mass spectrometry identified the primary cleavage sites to be K13 and K17 in histatin 1, R22, Y24, and R25 in histatin 3, and Y10, K11, R12, K13, H15, K17, H18 in histatin 5. Conclusion: The catalytic efficiency of histatin degradation by human saliva proteases is histatin 5>histatin 3> histatin 1. Proteolysis of histatins in human saliva is a stepwise process with particular histatin-dependent cleavage site specificity despite their structural similarities. This study was supported by NIH/NIDCR grants DE05672, DE07652, DE14950, and DE16699.