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 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 alpha-helical structure in a hydrophobic environment. Additionally, the circular dichroism data showed that Hsn-5 in an alpha-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.
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Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, 14214, USA.
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Bobek
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D000704:Analysis of Variance; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000737:chemistry* / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D054808:Histatins; D006801:Humans; D008561:Membrane Fusion; D010455:Peptides; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D013172:Spores, Fungal / Q000187:drug effects
D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; C068075:HTN3 protein, human; D054808:Histatins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides