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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.

REFERENCES

• Atherton E., Sheppard RC (1990). In: Solid phase peptide synthesis: a practical approach. Rickwood D, Hames BD, editors. Oxford, UK: IRL, pp. 131-148.
• Bairaktari E., Mierke DF, Mammi S., Peggion E. (1990). Conformation of bombolitins I and III in aqueous solutions: circular dichroism, 1H NMR, and computer simulation studies. Biochemisty 29:10097-10102.
• Baum BJ, Bird JL, Longton RW (1977). Polyacrylamide gel electrophoresis of human salivary histidine-rich polypeptide. J Dent Res 56:1115-1118.
• Bello J., Bello HR, Granados E. (1982). Conformation and aggregation of melittin: dependence on pH and concentration. Biochemistry 21:461-465.[CrossRef][Medline] [Order article via Infotrieve]
• Cassim JY, Yang JT ( 1967). Effect of molecular aggregation on circular dichroism and optical rotation dispersion of helical poly-L-glutamic acid in solution. Biochem Biophys Res Commun 26:58-64.[Medline] [Order article via Infotrieve]
• Cruciani RA, Barker JL, Durell SR, Raghunathan G., Guy HR, Zasloff M., et al. (1992). Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes. Eur J Pharmacol 226:287-296.[CrossRef][Medline] [Order article via Infotrieve]
• Edgerton M., Koshlukova SE, Lo TE, Chrzan BG, Straubinger RM, Raj PA (1998). Candidacidal activity of salivary histatins. J Biol Chem 273:20438-20447.[Abstract/Free Full Text]
• Gesell J., Zasloff M., Opella SJ (1997). Two-dimensional 1H NMR experiments show that the 23-residue magainin antibiotic peptide is an alpha-helix in dodecylphosphocholine micelles, sodium dodecylsulfate micelles, and trifluoroethanol/water solution. J Biomol NMR 9:127-135.[CrossRef][Medline] [Order article via Infotrieve]
• Greenfield N., Fasman GD (1969). Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8:4108-4116.[CrossRef][Medline] [Order article via Infotrieve]
• Helmerhorst EJ, van 't Hof W., Veerman EC, Simmons-Smit I., Nieuw Amerongen AV (1997). Synthetic histatin analogues with broad-spectrum antimicrobial activity. Biochem J 326:39-45.[Medline] [Order article via Infotrieve]
• Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61-68.[CrossRef][Medline] [Order article via Infotrieve]
• Jung G., Dubischar N. (1975). Conformational changes of alamethicin induced by solvent and temperature: a 13C-NMR and circular-dichroism study. Eur J Biochem 54:395-409.[Medline] [Order article via Infotrieve]
• Jung G., Bruckner H., Schmitt H. (1981). Properties of the membrane modifying polypeptide antibiotics alamethicin and trichotoxin A-40. In: Structure and activity of natural peptides. Voelter W, Weitzel G, editors. Berlin: Walter de Gruyter, pp. 75-114.
• Kiyota T., Lee S., Sugihara G. (1996). Design and synthesis of amphiphilic a-helical model peptides with systematically varied hydrophobic-hydrophilic balance and their interaction with lipid- and bio-membrane. Biochemistry 35:13196-13204.[CrossRef][Medline] [Order article via Infotrieve]
• Laemmli UK (1970). Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685[CrossRef][Medline] [Order article via Infotrieve]
• Lal K., Santarpia RP III, Xu L., Manssuri F., Pollock JJ (1992). One-step purification of histidine-rich polypeptides from human parotid saliva and determination of anti-candidal activity. Oral Microbiol Immunol 7:44-50.[Medline] [Order article via Infotrieve]
• Lear JD, Wasserman ZR, DeGrado WF (1988). Synthetic amphiphilic peptide models for protein ion channels. Science 240:1177-1181.[Abstract/Free Full Text]
• Mayr W., Oekonomopulos R., Jung G. ( 1979). Synthesis and conformation of a polyoxyethylene-bound undecapeptide of the alamethicin helix and (2-methylalanyl-L-alanine). Biopolymers 18:425-450.
• Oekonomopulos R., Jung G. ( 1980). Circular dichroism and conformational analysis of the membrane-modifying peptide N-t-Boc-(Aib-L-Ala)5-Gly-Ala-Aib-Pro-Ala-Aib-Aib-Glu-(OBzl)-Gln-OMe with respect to alamethicin. Biopolymers 19:203-214.[Medline] [Order article via Infotrieve]
• Oppenheim FG (1989). Salivary histidine-rich proteins. In: Human saliva: clinical chemistry and microbiology. Tenovuo JO, editor. Boca Raton, FL: CRC, pp. 151-160.
• Pollock JJ, Denepitiya L., Mackay BJ, Iacono VJ (1984). Fungistatic and fungicidal activity of human parotid salivary histidine-rich polypeptides on Candida albicans. Infect Immun 44:702-707.[Abstract/Free Full Text]
• Raj PA, Edgerton M., Levine MJ (1990). Salivary histatin-5: dependence of sequence, chain length, and helical conformation for candidacidal activity. J Biol Chem 265:3898-3905.[Abstract/Free Full Text]
• Raj PA, Johnsson M., Levine MJ, Nancollas GH (1992). Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization. J Biol Chem 267:5968-5976.[Abstract/Free Full Text]
• Raj PA, Soni SD, Levine MJ (1994). Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins. J Biol Chem 269:9610-9619.[Abstract/Free Full Text]
• Raj PA, Marcus E., Sukumaran DK (1998). Structure of human salivary histatin 5 in aqueous and nonaqueous solutions. Biopolymers 45:51-67.[CrossRef][Medline] [Order article via Infotrieve]
• Ramalingam K., Ramasubbu N., Levine MJ (1996). Effect of acetylation and permethylation on the conformation and candidacidal activity of salivary histatin-5. Lett Pept Sci 3:349-356.
• Sanger F., Nicklen S., Coulson AR (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467.[Abstract/Free Full Text]
• Sudha TS, Vijayakumar EKS, Balaram P. (1983). Circular dichroism studies of helical oligopeptides. Int J Peptide Protein Res 22:464-468.[Medline] [Order article via Infotrieve]
• Tsai H., Bobek LA (1997). Studies of the mechanism of human salivary histatin-5 candidacidal activity with histatin-5 variants and azole-sensitive and -resistant Candida species. Antimicrob Agents Chemother 41:2224-2228.[Abstract]
• Tsai H., Bobek LA (1998). Human salivary histatins: promising anti-fungal therapeutic agents. Crit Rev Oral Biol Med 9:480-497.[Abstract/Free Full Text]
• Tsai H., Raj PA, Bobek LA (1996). Candidacidal activity of recombinant human salivary histatin-5 and variants. Infect Immun 64:5000-5007.[Abstract]
• Tsai H., Raj PA, Bobek LA (1997). Candidacidal activity and helical conformation of active C-terminal fragments of human salivary histatin-5. Int J Oral Biol 22:67-71.
• Vijayakumar EK, Crisma M., Toniolo M., Balaram P. (1983). Stereochemistry of alpha-aminoisobutyric acid peptides in solution: helical conformation of protected decapeptides with repeated Aib-L-Ala and Aib-L-Val sequence. Biopolymers 22:2133-2140.[Medline] [Order article via Infotrieve]
• Voet D., Voet J. ( 1995). Biochemistry. 2nd ed. New York: John Wiley & Sons.
• Woolley GA, Wallace BA (1992). Model ion channels: gramicidin and alamethicin. J Membrane Biol 129:109-136.[Medline] [Order article via Infotrieve]
• Xu T., Levitz SM, Diamond RD, Oppenheim FG (1991). Anticandidal activity of major human salivary histatins. Infect Immun 59:2549-2554.[Abstract/Free Full Text]
• Zuo Y., Xu T., Troxler RF, Li J., Driscoll J., Oppenheim FG (1995). Recombinant histatins: functional domain duplication enhances candidacidal activity. Gene 161:87-91.[CrossRef][Medline] [Order article via Infotrieve]

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


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