Sage Journals: Discover world-class research

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

Get full access to this article

View all access options for this article.

References

Baum BJ , Bird JL , Millar DB , Longton RW (1976). Studies on histidine-rich polypeptides from human parotid saliva. Arch Biochem Biophys 177:427-436.

Bobek LA , Wang X. , Levine MJ (1993). Efficient production of biologically active human salivary cystatins in Escherichia coli. Gene 123:203-210.

Bobek LA , Ramasubbu N. , Wang X. , Weaver TR , Levine MJ (1994). Biological activities and secondary structures of variant forms of human salivary cystatin SN produced in Escherichia coli . Gene 151:303-308.

Dabbagh WK , Xu T. , Oppenheim FG (1994). Immunological and densitometric determination of histatin concentration in saliva (abstract). J Dent Res 73:150.

Dawes C. , Jenkins GN , Tonge CH (1963). The nomenclature of the integuments of the enamel surface of teeth. Br Dent J 115:65-68.

Hay DI (1973). The interaction of human parotid salivary proteins with hydroxyapatite. Arch Oral Biol 18:1517-1529. Hay DI (1975). Fractionation of human parotid salivary proteins and the isolation of a histidine-rich acidic peptide which shows high affinity for hydroxyapatite surfaces. Arch Oral Biol 20:553-558.

Hay DI , Moreno EC , Schlesinger DH (1979). Phosphoprotein inhibitors of calcium phosphate precipitation from salivary secretions. Inorg Persp Biol Med 2:271-285.

Hay DI , Bennick A. , Schlesinger DH , Minaguchi K. , Madapallimattam G. , Schluckebier SK (1988). The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f). Biochem J 255:15-21.

Jensen JL , Lamkin MS , Oppenheim FG (1992). Adsorption of human salivary proteins to hydroxyapatite: A comparison between whole saliva and glandular salivary secretions. J Dent Res 71:1569-1576.

10.

Johnsson M. , Richardson CF , Bergey EJ , Levine MJ , Nancollas GH (1991). The effects of human salivary cystatins and statherin on hydroxyapatite crystallization. Arch Oral Biol 36:631-636.

11.

Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 :680-685.

12.

Lal K. , Xu L. , Colburn J. , Hong AL , Pollock JJ (1992). The use of capillary electrophoresis to identify cationic proteins in human parotid saliva. Arch Oral Biol 37:7-13.

13.

Lamkin M. , Oppenheim FG (1993). Structural features of salivary function. Crit Rev Oral Biol Med 4:251-259.

14.

MacKay BJ , Pollock JJ , Iacono VJ , Baum BJ ( 1984a). Isolation of milligram quantities of a group of histidine-rich polypeptides from human parotid saliva. Infect Immun 44:688-694.

15.

MacKay BJ , Denepitiya L. , Iacono VJ , Krost SB , Pollock JJ (1984b). Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans . Infect Immun 44:695-701.

16.

Mayhall CW (1970). Concerning the composition and source of the acquired enamel pellicle of human teeth. Arch Oral Biol 15:1327-1341.

17.

Moreno E. , Kresak M. , Hay DI (1978). Adsorption of two human parotid salivary macromolecules on hydroxy-, fluorohydroxy and fluoroapatites. Arch Oral Biol 23 :525-533.

18.

Odds FC ( 1980). A simple system for the presumptive identification of Candida albicans and differentiation of strains within the species. Sabouraudia 18:301-317. Oppenheim FG (1989). Cationic salivary proteins. In: Clinical chemistry of human saliva. Salivary histidine-rich proteins. Vol. 1. Tenuovo J, editor. Boca Raton, FL: CRC Press, pp. 151-160.

19.

Oppenheim FG , Offner GD , Troxler RF (1982). Phosphoproteins in the parotid saliva from the subhuman primate Macaca fascicularis. J Biol Chem 257:9271-9282.

20.

Oppenheim FG , Offner GD , Troxler RF (1985). Amino acid sequence of proline-rich phosphoglycoprotein from parotid secretion of the subhuman primate Macaca fascicularis. J Biol Chem 260:10671-10679.

21.

Oppenheim FG , Yang YC , Diamond RD , Hyslop D. , Offner GD , Troxler RF (1986). The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion. J Biol Chem 261:1177-1182. Oppenheim FG , Xu T. , McMillian FM , Levitz SM , Diamond RD , Offner GD , et al. (1988). Histatins, a novel family of histidine-rich proteins in human parotid secretion . J Biol Chem 263:7472-7477.

22.

Perlman GE (1952). The phosphorus in pepsin and pepsinogen.

23.

J Am Chem Soc 74:6308-6312.

24.

Pollock JJ , Denepitiya L. , MacKay BJ , Iacono VJ (1984). Fungistatic and fungicidal activity of human parotid salivary histidine-rich polypeptides on Candida albicans. Infect Immun 44:702-707.

25.

Raj PA , Edgerton M. , Levine MJ (1990). Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity. J Biol Chem 265:3898-3905.

26.

Sabatini LM , Azen EA ( 1989). Histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (His1 and His2). Biochem Biophys Res Commun 160:495-502. Schlesinger DH , Hay DI (1977). Complete covalent structure of statherin, a tyrosine-rich acidic protein which inhibits calcium phosphate precipitation from human parotid saliva. J Biol Chem 252:1689-1695.

27.

Schlesinger DH , Buku A. , Wyssbrod HR , Hay DI (1987). Chemical synthesis of phosphoseryl-phosphoserine, a partial analogue of human salivary statherin, a protein inhibitor of calcium phosphate precipitation in human saliva. Int J Pept Prot Res 30:257-262.

28.

Shomers JP , Tabak LA , Levine MJ , Mandel ID , Ellison SA (1982). The isolation of a family of cysteine-containing phosphoproteins from submandibular-sublingual saliva. J Dent Res 61:973-977.

29.

Smith DB , Johnson KS (1988). Single-step purification of peptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40.

30.

Sugiyama K. (1993). Anti-lipopolysaccharide activity of histatins, peptides from human saliva. Experientia 49:1095-1097. Svanborg A. , Svennerholm L. (1961). Plasma total lipid, cholesterol, triglycerides, phospholipids and free fatty acids in a healthy Scandinavian population. Acta Med Scand 169:43-49.

31.

Troxler RF , Offner GD , Xu T. , vanderSpek JC , Oppenheim FG (1990). Structural relationship between human salivary histatins. J Dent Res 69:2-6.

32.

Tsai H. , Levine MJ , Bobek LA (1993). Purification and biological activities of recombinant human salivary cystatin/histatin fusion protein (abstract). J Dent Res 72:209.

33.

vanderSpek JC , Wyandt HE , Skare JC , Milunsky A. , Oppenheim FG , Troxler RF (1989). Localization of the genes for histatins to human chromosome 4ql3 and tissue distribution of the mRNAs. Am J Hum Genet 45:381-387. Wong RSC , Hofmann T. , Bennick A. (1979). The complete primary structure of a proline-rich phosphoprotein from human saliva. J Biol Chem 254:4800-4808.

34.

Xu T. , Oppenheim FG (1993). Salivary antimicrobials: Where are we? In: Cariology for the nineties. Bowen WH , Tabak LA , editors. Rochester, NY: University of Rochester Press, pp. 117-131.

35.

Xu T. , Levitz SM , Diamond RD , Oppenheim FG (1991). Anticandidal activity of major human salivary histatins. Infect Immun 59:2549-2554.

Functional Comparison of Native and Recombinant Human Salivary Histatin 1

Abstract

Keywords

Get full access to this article

References