title: Anti-candidal activity of genetically engineered histatin variants with multiple functional domains.
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.
authors.affiliation: Department of Periodontology and Oral Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA. fropp@bu.edu
authors.forename: Frank G
authors.initials: FG
authors.lastname: Oppenheim
authors.forename: Eva J
authors.initials: EJ
authors.lastname: Helmerhorst
authors.forename: Urs
authors.initials: U
authors.lastname: Lendenmann
authors.forename: Gwynneth D
authors.initials: GD
authors.lastname: Offner
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D002175:Candida / Q000166:cytology / Q000187:drug effects*; D005818:Genetic Engineering; D054808:Histatins / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors
chemical_list: D000935:Antifungal Agents; D054808:Histatins; D050505:Mutant Proteins; D011994:Recombinant Proteins