| dc.publisher | Boston>Three major histatins, histatins 1, 3 and 5, from human parotid secretion were isolated by gel filtration and reversed-phase high-performance liquid chromatography. The complete amino acid sequences of histatins were determined by automated Edman degradation of the protein, Staphylococcus aureus V8 protease, and tryptic peptides, are as follows:
Histatin 1: NH[2]-Asp-Pse-His-Glu-Lys-Arg-His-His-Gly-Tyr-Arg--Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Tyr-Gly-Ser-Asn-Tyr-Leu-Tyr-Asp-Asn-COOH
Histatin 3: NH[2]-Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr-Arg-Ser-Asn-Tyr-Leu-Tyr-Asp-Asn-COOH
Histatin 5: NH[2]-Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr-COOH
Histatins 1, 3, and 5 contain 38, 32, and 24 amino acid residues have molecular weights of 4929, 4063, and 3037, respectively. Histatin 1 contains one mole of phosphate per mole of protein. Histatins 3 and 5 lack phosphate. With the exception of Glu (residue 4) and Arg (residue 11) in histatin 1, the first 22 amino acid residues of three histatins are identical, and the carboxyl-terminal 7 residues of histatin 1 and 3 are also identical. The sequence, -Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Try-GIy- (residues 23-31), in histatin 1 is absent in histatin 1. The complete sequence of histatin 5 is contained within the amino termina1 24 residues of histatin 3.
For the functional characterization, three bioassays were designed to investigate the antimicrobial property of histatins on Candida albicans, a dimorphic fungal pathogen. The three bioassays are: 1) killing of blastoconidium, 2) killing of germinated cell, and 3) inhibition of germination. Candidacidal activities were further evaluated at different ionic concentrations, in the presence of assorted mono- and divalent ions, and at various pH values. In addition, the susceptibility of C. albicans in different growth phases to histatins was investigated. While all three major histatins demonstrated candidacidal activities, they differed in their abilites to kill blastoconidium and germ tubes with histatin 5 being the most active, histatin 3 showing moderate activity and histatin 1 exhibiting the lowest level of activity. For the inhibition of germination, however, histatin 3 exhibited more activity than either histatins l and 5.
The candidacidal activity of histatins was inversely proportional to both ionic strength and divalent cation concentration in the medium. Stepwise reduction of the pH of the assay medium enhanced the candidacidal activities of histatins 1 and 3, while the activity of histatin 5 was pH independent over the range of pH 4-8.C. albicans in log phase growth was more susceptible to histatins 1 and 3 than cells in stationary phase. Cells in either growth phase were still more vulnerable to histatin 5 than to histatins 1 and 3. The results obtained establish the functional relationship of the major histatins with respect to both their fungicidal and fungistatic activities and provide insights into their activities at ionic and pH conditions likely to be encountered in vivo in the oral cavity. Moreover, the data point towards possible mechanisms responsible for the anticandidal activities of histatins. | en_US |
