Functional comparison of native and recombinant human salivary histatin 1.
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-Hst1) 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-Hst1 fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hst1 from the GST fusion partner. The digest was subjected to reversed-phase high-performance liquid chromatography on a C18 column, and re-Hst1 was eluted as a well-defined peak. The yield of re-Hst1 was 4 mg/L of bacterial culture. Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hst1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hst1 had the same apparent molecular weights, while cationic PAGE showed that re-Hst1 was more basic. Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hst1 lacked any detectable phosphate. Re-Hst1 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.
Driscoll
J
J
Department of Periodontology and Oral Biology, School of Graduate Dentistry, Boston University Medical Center, MA 02118, USA.
Zuo
Y
Y
Xu
T
T
Choi
J R
JR
Troxler
R F
RF
Oppenheim
F G
FG
D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D002176:Candida albicans / Q000187:drug effects; D003001:Cloning, Molecular; D017931:DNA Primers; D017886:Durapatite / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D054808:Histatins; D006801:Humans; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D000935:Antifungal Agents; D017931:DNA Primers; D054808:Histatins; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides; C068074:HTN1 protein, human; D010768:Phosphoserine; D017886:Durapatite