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Construction and Characterization of Human Salivary Histatin-5 Multimers

Department of Oral Biology, 109 Foster Hall, School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York 14214

H. Tsai

Department of Oral Biology, 109 Foster Hall, School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York 14214

L.A. Bobek

Department of Oral Biology, 109 Foster Hall, School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York 14214

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 hypotheses: 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 like other cationic peptides which aggregate and form channels across the target membrane. A PCR-based gene splicing by overlap extension (SOE) method was used to construct the DNA fragments encoding the following fusion molecules: 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 activities as well as secondary structures were determined. Contrary to our hypotheses, results showed that none of the multimers possessed increased candidacidal activity. Specifically, C16—C16 and Hsn-5—C16 displayed candidacidal activity comparable with that of Hsn-5, while Hsn-5-Hsn-5 possessed significantly 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 a-helical conformation does not aggregate in a hydrophobic environment, not even at 14- to 18-fold its physiological concentration. Our results suggest that the development of enhanced Hsn-5 molecules may not be achieved by duplication of its functional domain, and that Hsns may not act like other antimicrobial cationic peptides which aggregate and form channels across the target membrane.

Journal of Dental Research, Vol. 78, No. 2, 690-698 (1999)
DOI: 10.1177/00220345990780020901


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