Construction and characterization of human salivary histatin-5 multimers

Human salivary histatin-5 (Hsn-5), a 24-amino acid polypeptide, is a potent candidacidal molecule. In this study, we have explored the following two h ypotheses: More potent Hsn molecules may be achieved by duplication of the functional domain of Hsn-5 (C16, residues 9-24 of Hsn-5), and Hsn may act l ike other cationic peptides which aggregate and form channels across the ta rget membrane. A PCR-based gene splicing by overlap extension (SOE) method was used to construct the DNA fragments encoding the following fusion molec ules: Hsn-5-Hsn-5, Hsn-5-C16, and C16-C16. These constructs were expressed in E. coli, the proteins produced were purified, and their anticandidal act ivities as well as secondary structures were determined. Contrary to our hy potheses, results showed that none of the multimers possessed increased can didacidal activity. Specifically, C16-C16 and Hsn-5-C16 displayed candidaci dal activity comparable with that of Hsn-5, while Hsn-5-Hsn-5 possessed sig nificantly decreased candidacidal activity, yet all molecules retained an a -helical structure in a hydrophobic environment. Additionally, the circular dichroism data showed that Hsn-5 in an or-helical conformation does not ag gregate in a hydrophobic environment, not even at 14- to 18-fold its physio logical concentration. Our results suggest that the development of enhanced Hsn-5 molecules may not be achieved by duplication of its functional domai n, and that Hsns may not act like other antimicrobial cationic peptides whi ch aggregate and form channels across the target membrane.