Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates, This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC(50)s) of 0.57 and 0.25 muM , respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 muM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9, These results point to a functional domain localized in the C-terminal part of histatin 5, To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas giltgivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the K-m with a K-i of 15 muM. In contrast, inhibition of Lys-gingipain affected both the K-m and V-max, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity.