Functional Comparison ofNative andRecombinant HumanSalivary Histatin 1

Histatin 1isahistidine-rich phosphoprotein present inhumanparotid saliva that possesses candidacidal activity andfunctions inmineralization byadsorbing to hydroxyapatite. Theobjective ofthepresent studywasto develop asystem forrecombinant production ofhistatin 1 andtoexamine therole ofphosphorylation inthefunctional activities ofthis molecule. Native histatin 1(containing a phosphoserine atresidue 2)waspurified fromparotid saliva, whereas abacterial expression system wasusedto produce arecombinant formofhistatin 1(re-Hstl) that lacked phosphorylated serine. Histatin 1cDNA was inserted intothevector pGEX-3X, whichexpresses foreign genesassoluble fusion proteins attached tothecarboxyl- terminus ofglutathione S-transferase (GST). TheGST/re- Hstlfusion protein wasisolated fromcell lysates byaffinity chromatography onglutathione (GSH)-Sepharose and digested withcyanogen bromide toseparate re-Hstl from theGSTfusion partner. Thedigest wassubjected to reversed-phase high-performance liquid chromatography onaC18column, andre-Hstl waseluted asawell-defined peak. Theyield ofre-Hstl was4mg/Lofbacterial culture. Amino-terminal sequencing andaminoacidanalysis confirmed thefinal product asre-Hstl. Sodiumdodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) showedthatnative histatin 1andre-Hstl hadthesame apparent molecular weights, while cationic PAGEshowed that re-Hstl wasmorebasic. Phosphate analysis indicated 1 molphosphate/mol ofnative histatin 1,whilere-Hstl lacked anydetectable phosphate. Re-Hstl demonstrated candidacidal activity comparable tothat ofnative histatin 1, but displayedsubstantially lowerbindingto hydroxyapatite. Theseresults showthatphosphorylation of histatin 1atresidue 2contributes significantly toitsability tobindtohydroxyapatite.