Oral fluid proteolytic effects on histatin 5 structure and function E.J. Helmerhorst * , A.S. Alagl 1 , W.L. Siqueira, F.G. Oppenheim Department of Periodontology and Oral Biology, Boston University, Goldman School of Dental Medicine, 700 Albany Street, Boston, MA 02118, United States Accepted 12 June 2006 Introduction The parotid and submandibular/sublingual glands are the main contributors to oral fluid, more commonly called whole saliva. These glands secrete a wide variety of proteins, ranging in size from 3 kD to over 100 kD. Their functions are of critical importance in Archives of Oral Biology (2006) 51, 1061—1070 www.intl.elsevierhealth.com/journals/arob KEYWORDS Saliva proteolysis; Histatins; Degradation; Oral; Antifungal Summary 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 pro- ducts 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 Æ 22 mg/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 repre- sents 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. # 2006 Elsevier Ltd. All rights reserved. Abbreviations: CHCA, alpha-cyano-4-hydroxy-cinnamic acid; kD, kilo Daltons; MALDI-TOF, matrix-assisted laser desorption/ ionisation-time of flight; OD, optical density; RP-HPLC, reversed- phase high performance liquid chromatography; SDB, Sabouraud dextrose broth; WSS, whole saliva supernatant * Corresponding author. Tel.: +1 617 414 1119; fax: +1 617 638 4924. E-mail address: helmer@bu.edu (E.J. Helmerhorst). 1 Present address: College of Dentistry, King Faisal University, P.O. Box 40037, Alkhobar 31952, Saudi Arabia. 0003–9969/$ — see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2006.06.005