Abstract
The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins.
Highlights
In the last decades, a number of cationic proteins have been identified which have molecular weights ranging between 2,000 to 6,000 Da and potent antimicrobial activities [1,2,3]
Generation of Histatin Multimers DNA fragments encoding recombinant histatin 3 with one, two, three, or four functional domains were generated in sequential PCR reactions using overlap extension [24]
This resulted in a recombinant histatin 3 fragment with two functional domains
Summary
A number of cationic proteins have been identified which have molecular weights ranging between 2,000 to 6,000 Da and potent antimicrobial activities [1,2,3]. The antifungal domain of histatins has been localized to a 1,875 Da domain containing 14 amino acid residues in the middle region of histatin 3 [9,10,11] This domain is highly conserved in the three major human histatins, and can be found in histatins from the subhuman primate Macaca fascicularis [12]. It comprises amino acid residues 12–25 of histatin 3 (RKFHEKHHSHRGYR) and exhibits an LC50 of 0.3 mM which surpasses the killing activity of all native histatins [8]. Of clinical importance is the potential exploitation of such functional bioactive peptides for therapeutic purpose [16]
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