title: Oral fluid proteolytic effects on histatin 5 structure and function.
abstract: Histatins are human salivary antifungal proteins that are prone to extensive enzymatic degradation upon their release into the oral cavity. Histatin proteolysis, leading to the disappearance of the intact protein can be expected to have functional consequences. Histatin 5, comprising 24 residues, is the smallest of the major salivary histatins and the most active in terms of its antifungal properties. The rate and mode of histatin 5 degradation were determined by incubating the protein in whole saliva supernatant for various time intervals. Fragmentation products were collected by reversed-phase high performance liquid chromatography (RP-HPLC), characterised structurally by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and functionally in a fungal growth inhibition assay. Of the 19 fragments identified, 16 were derived from single proteolytic cleavage events in histatin 5. A remarkable finding was the inter-subject consistency in the histatin 5 degradation pattern. Added histatin 5 disappeared from whole saliva supernatant at an average rate of 105+/-22 microg/ml/h, which in part could explain the virtual absence of histatin 5 in whole saliva. Despite the rapid proteolysis of histatin 5, the early degradation mixture was as active in antifungal assays as intact histatin 5. These data demonstrate that the oral-fluid mediated proteolysis of histatin 5 represents an intrinsic biological property of whole saliva. The data also reveal that the early proteolysis phase of histatin 5 does not abolish the antifungal properties associated with this protein.
authors.affiliation: Department of Periodontology and Oral Biology, Boston University, Goldman School of Dental Medicine, 700 Albany Street, Boston, MA 02118, USA. helmer@bu.edu
authors.forename: E J
authors.initials: EJ
authors.lastname: Helmerhorst
authors.forename: A S
authors.initials: AS
authors.lastname: Alagl
authors.forename: W L
authors.initials: WL
authors.lastname: Siqueira
authors.forename: F G
authors.initials: FG
authors.lastname: Oppenheim
mesh_terms: D000328:Adult; D000935:Antifungal Agents / Q000378:metabolism*; D002176:Candida albicans / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D054808:Histatins; D006801:Humans; D009055:Mouth / Q000382:microbiology; D010446:Peptide Fragments / Q000032:analysis; D011480:Protease Inhibitors / Q000378:metabolism*; D011489:Protein Denaturation; D011506:Proteins / Q000378:metabolism / Q000502:physiology*; D015203:Reproducibility of Results; D012463:Saliva / Q000201:enzymology / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000378:metabolism / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D000935:Antifungal Agents; C068075:HTN3 protein, human; D054808:Histatins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011506:Proteins; D012471:Salivary Proteins and Peptides