Oral Fluid Proteolysis and its Effect on Histatin 5

Introduction: Histatins are human salivary antifungal proteins containing multiple potential cleavage sites for trypsin and chymotrypsin, enzymatic activities that are well known to be present in whole saliva (WS). This likely explains why histatin concentrations in WS are twenty- to forty-fold lower than those in the major salivary secretions. Objective: To determine the primary cleavage sites in histatin 5 in WS supernatant (WSS) obtained from healthy donors. Methods: Pure histatin 5 (400 µg/ml) was added to 1:10 or 1:100 dilutions of WSS from one donor or a pool of WSS derived from six other donors. After various incubation times, 100 µl aliquots were removed, boiled and subjected to RP-HPLC. Histatin peptides generated by degradation were collected, subjected to MALDI-TOF mass spectrometric analysis, and the m/z values were compared to masses from a theoretical histatin fragmentation data base. Results: Seventeen fragments representing the early degradation products of histatin 5 were defined in terms of their amino acid sequence. The cleavage sites uncovered allowed the determination of the type of enzymatic activity which is present in WSS and is responsible for histatin 5 degradation upon release into the oral cavity. The degradation pattern observed with WSS from one subject and that obtained with the pool of WSS from six subjects was qualitatively identical suggesting inter-individual consistency of histatin degradation. Conclusion: The identification of the initial degradation products of histatin 5 and the known antimicrobial activity of at least some of these fragments suggests that considerable functional capacity may be retained during the early phases of histatin degradation. This study was supported by NIH/NIDCR Grants DE05672, DE07652 and DE14950.