Functional Comparison of Native and Recombinant Human Salivary Histatin 1

Abstract

Histatin 1 is a histidine-rich phosphoprotein present in human parotid saliva that possesses candidacidal activity and functions in mineralization by adsorbing to hydroxyapatite. The objective of the present study was to develop a system for recombinant production of histatin 1 and to examine the role of phosphorylation in the functional activities of this molecule. Native histatin 1 (containing a phosphoserine at residue 2) was purified from parotid saliva, whereas a bacterial expression system was used to produce a recombinant form of histatin 1 (re-Hstl) that lacked phosphorylated serine. Histatin 1 cDNA was inserted into the vector pGEX-3X, which expresses foreign genes as soluble fusion proteins attached to the carboxyl-terminus of glutathione S-transferase (GST). The GST/re-Hstl fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hstl from the GST fusion partner. The digest was subjected to reversed-phase high-performance liquid chromatography on a C₁₈ column, and re-Hstl was eluted as a well-defined peak. The yield of re-Hstl was 4 mg/L of bacterial culture. Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hstl. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hstl had the same apparent molecular weights, while cationic PAGE showed that re-Hstl was more basic. Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hstl lacked any detectable phosphate. Re-Hstl demonstrated candidacidal activity comparable to that of native histatin 1, but displayed substantially lower binding to hydroxyapatite. These results show that phosphorylation of histatin 1 at residue 2 contributes significantly to its ability to bind to hydroxyapatite.

Keywords

histatin pGEX expression system glutathione-Sepharose phosphoprotein candidacidal hydroxyapatite binding

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