Candidacidal activity prompted by N-terminus histatin-like domain of human salivary mucin (MUC7) 1 Tarikere L. Gururaja 2 , Joseph H. Levine, Duy T. Tran, Gowda A. Naganagowda, Kalaiyarasi Ramalingam, Narayanan Ramasubbu, Michael J. Levine * Department of Oral Biology and Dental Research Institute, State University of New York at Bu¡alo, 109 Foster Hall, 3435 Main Street, Bu¡alo, NY 14214, USA Received 7 January 1999; accepted 25 January 1999 Abstract Histidine-rich peptides (histatins, Hsn) in saliva are thought to provide a non-immune defense against Candida albicans. Sequence homology search of the human salivary mucin, MUC7, against histatins revealed a domain at the N-terminus (R 3 ^ Q 17 ) having 53% identity to Hsn-5. To determine its candidacidal activity, this 15 residue basic histidine-rich domain of MUC7 (I) was prepared by solid-phase Fmoc chemistry. Various N- and C-terminal protected derivatives of I were also synthesized to correlate the effect of peptide overall charge in exhibiting cidal potency. Candidacidal activity measurement of I and its variants showed considerable ED 50 values (effective dosage required to kill 50% of candida cells), albeit greater than Hsn-5 (ED 50 V4^6 WM). Of the various analogs tested, N-terminal free acid (I, ED 50 V40 WM) and amide (V, ED 50 V16 WM) exhibited appreciable candidacidal activities suggesting the possible role of peptide net charge in cidal action. Blocking of N-terminus with a bulky octanoyl group showed only marginal effect on the cidal activity of I or V, indicating that hydrophobicity of these synthetic constructs may not be important for exerting such activities. Membrane-induced conformational transition from random coil to helical structures of all the test peptides implied their tendency to adapt order structures at the lipid-membrane interface similar to that of Hsn-5. However, comparison of propensity for helical structure formation vs. ED 50 indicated that cidal potency of MUC7 Hsn-like peptides depends largely on electrostatic interactions irrespective of secondary structural elements. Delineation of solution structure of the most active peptide (V) by 2D-NMR revealed essentially a non-structured conformation in aqueous medium, which further supported the fact that the peptide helical structure may not be a prerequisite for posing candidacidal activity. The formation of smaller truncated peptides 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4838(99)00034-5 Abbreviations : Abbreviations for amino acids follow the recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature in Eur. J. Biochem. 138 (1984) 9^37; aa, amino acid; CD, circular dichroism; CSI, chemical shift index; DQF-COSY, double quantum ¢ltered correlated spectroscopy ; Fmoc, N K -£uorenylmethoxycarbonyl ; HPLC, high-performance liquid chromatogra- phy ; Hsn, histatins ; 1D- and 2D-NMR, one- and two-dimensional nuclear magnetic resonance spectroscopy ; MALDI, matrix-assisted laser desorption/ionization ; NOESY, nuclear Overhauser enhancement spectroscopy ; PAGE, polyacrylamide gel electrophoresis ; ROESY, rotating frame Overhauser enhancement spectroscopy ; SAR, structure-activity relationships ; SDS, sodium dodecyl sulfate ; SPPS, solid-phase peptide synthesis ; TBS, Tris-bu¡ered saline ; TOCSY, total correlation spectroscopy * Corresponding author. Fax : +1 (716) 829-3942 ; E-mail : mj_levine@sdm.bu¡alo.edu 1 Supplementary material : complete 1 H-NMR chemical shift data of the peptide, V, in various solvents obtained in this study have been deposited with the BioMagResBank Data Center (http://www.bmrb.wisc.edu) and the data can be retrieved with the accession number 4273. Additional information can be obtained upon request to Dr. Eldon Ulrich, Director, BMRB, University of Wisconsin, Madison, USA (Fax: +1 (608) 262-3453 oe e-mail: elu@bmrb.wisc.edu). 2 Present address : Rigel Pharmaceuticals, 772 Lucerne Drive, Sunnyvale, CA 94086, USA. Biochimica et Biophysica Acta 1431 (1999) 107^119