Pellicle Precursor Protein Crosslinking: Characterization of an Adduct between Acidic Proline-rich Protein (PRP-1) and Statherin Generated by Transglutaminase

Abstract

Recent work with oral transglutaminase indicated that this enzyme, derived from oral epithelial cells, crosslinked pellicle precursor proteins which may be important in the formation of the acquired enamel pellicle. The purpose of this study was to investigate whether purified acidic PRP-1 can form crosslinks with statherin, and whether such a crosslink is derived from a transglutaminase-catalyzed reaction between glutaminyl and lysyl side-chains, leading to a covalent bond formation. Enzymatic reaction products were analyzed by SDS-PAGE and reverse-phase HPLC. The SDS electrophoretogram revealed a protein band with an apparent molecular weight of 32 kDa, which is consistent with the combined apparent molecular weight of acidic PRP-1 (24 kDa) and statherin (8 kDa). A reaction product isolated by HPLC was characterized by amino acid analysis, which showed a stoichiometry consistent with being an adduct composed of one molecule of acidic PRP-1 and one molecule of statherin. In negative control experiments, it could be shown that this adduct was not detected when the lysines of both substrates were modified by reductive methylation prior to the enzymatic reaction. In addition, amino acid analysis and mass spectrometry confirmed the presence of a γ-glutamyl-∈-lysine dipeptide after enzymatic hydrolysis and the absence of this dipeptide after acid hydrolysis. Analysis of the data obtained indicates that oral transglutaminase is capable of crosslinking acidic PRP-1 and statherin in vitro. In addition, this finding exemplifies the potential of post-secretory processing of salivary proteins, which may represent an additional mechanism to generate new protein species.

Keywords

acquired enamel pellicle transglutaminase salivary proteins.

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References

Barsigian C. , Fellin FM , Jain A. , Martinez J. (1988). Dissociation of fibrinogen and fibronectin binding from transglutaminase-mediated cross-linking at the hepatocyte surface. J Biol Chem 263:14015-14022.

Bennick A. , Connell GE (1971). Purification and partial characterization of four proteins from human parotid saliva. Biochem J 123:455-464.

Bennick A. , Cannon M. , Madapallimattam G. (1979). The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins. Biochem J 183:115-126.

Bennick A. , Chau G. , Goodlin R. , Abrams S. , Tustian D. , Madapallimattam G. (1983). The role of human salivary acidic proline-rich proteins in the formation of acquired dental pellicle in vivo and their fate after adsorption to the human enamel surface. Arch Oral Biol 28:19-27.

Birckbichler PJ , Upchurch HF , Patterson Mkj , Conway E. (1985). High-molecular-weight human epidermal transglutaminase . J Invest Dermatol 85:75-78.

Bleiweis AS , Oyston Pcf (1993). Adhesion and cohesion of plaque microflora: a function of microbial fimbriae or fimbrils? In: Cariology for the nineties. Bowen WH , Tabak LA , editors. New York: University of Rochester Press, pp. 287-300.

Bradway SD , Bergey EJ , Jones PC , Levine MJ (1989). Oral mucosal pellicle. Adsorption and transpeptidation of salivary components to buccal epithelial cells. Biochem J 261:887-896.

Bradway SD , Bergey EJ , Scannapieco FA , Ramasubbu N. , Zawacki S. , Levine MJ (1992). Formation of salivary-mucosal pellicle: the role of transglutaminase. Biochem J 284:557-564.

Chung SI (1972). Comparative studies on tissue transglutaminase and factor XIII. Ann NYAcad Sci 202:240-255.

10.

Clarke DD , Mycek MJ , Neidle A. , Waelsch H. (1959). The incorporation of amines into proteins. Arch Biochem Biophys 79:338-354.

11.

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

12.

Folk JE , Chung SI (1973). Molecular and catalytic properties of transglutaminases . Adv Enzymol Relat Areas Mol Biol 38:109-191.

13.

Gibbons RJ Hay DI , Cisar JO , Clark WB (1988). Adsorbed salivary proline-rich protein 1 and statherin: receptors for type 1 fimbriae of Actinomyces viscosus T14V-J1 on apatitic surfaces. Infect Immun 56:2990-2993.

14.

Goldsmith LA , Baden HP , Roth SI , Colman R. , Lee L. , Fleming B. (1974). Vertebral epidermal transamidases. Biochim Biophys Acta 351:113-125.

15.

Hay DI (1967). The adsorption of salivary proteins by hydroxyapatite and enamel. Arch Oral Biol 12:937-946.

16.

Hay DI (1969). Some observations on human saliva proteins and their role in the formation of the acquired enamel pellicle. J Dent Res 48:806-810.

17.

Hay DI (1973a). The interaction of human parotid salivary proteins with hydroxyapatite. Arch Oral Biol 18:1517-1529.

18.

Hay DI (1973b). The isolation from human parotid saliva of a tyrosine rich acidic peptide which exhibits high affinity for hydroxyapatite surfaces. Arch Oral Biol 18:1531-1541.

19.

Hay DI , Carlson ER , Schluckebier SK , Moreno EC , Schlesinger DH (1987). Inhibition of calcium phosphate precipitation by human salivary acidic proline-rich proteins: structure-activity relationships . Calcif Tissue Int 40:126-132.

20.

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.

21.

Jensen JL , Lamkin MS , Troxler RF , Oppenheim FG (1991). Multiple forms of statherin in human salivary secretions. Arch Oral Biol 36:529-534.

22.

Jentoft N. , Dearborn DG (1983). Protein labeling by reductive alkylation. Meth Enrymol 91:570-579.

23.

Kubilus J. , Baden HP (1984). Isopeptide bond formation in epidermis. Mol Cell Biochem 58:129-137.

24.

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

25.

Lamkin MS , Migliari D. , Troxler RF , Oppenheim FG (1995). Novel post-translational processing in whole saliva (abstract). J Dent Res 74(Spec Iss):85.

26.

Loewy AG (1984). The N∈-(γ-glutamic)lysine cross-link: method of analysis, occurrence in extracellular and cellular proteins. Meth Enrymol 107:241-257.

27.

Oppenheim FG , Hay DI , Franzblau C. (1971). Proline-rich proteins from human parotid saliva. 1. Isolation and partial characterization. Biochemistry 10:4233-4238.

28.

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.

29.

Oppenheim FG , Xu T. , McMillian FM , Levitz SM , Diamond RD , Offner GD , et al. (1988). Histatin, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J Biol Chem 263:7472-7477.

30.

Plapp BV , Moore S. , Stein WH (1971). Activity of bovine pancreatic deoxyribonuclease A with modified amino groups. J Biol Chem 246:939-945.

31.

Schlesinger DH , Hay DI (1977). Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva. J Biol Chem 252:1689-1695.

32.

Schroeder HE (1981). Discussion. In: Differentiation of human oral stratified epithelia. New York: S. Karger, pp. 208-230.

33.

Staab JF , Bradway SD , Fidel PL , Sundstrom P. (1999). Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwpl. Science 283:1535-1538.

34.

Ta BM , Gallagher GT , Chakravarty R. , Rice RH ( 1990). Keratinocyte transglutaminase in human skin and oral mucosa: cytoplasmic localization and uncoupling of differentiation markers . J Cell Sci 95:631-638.

35.

Yao Y. , Lamkin MS , Oppenheim FG (1998). Pellicle precursor protein modification by transglutaminase (abstract). J Dent Res 77(Spec Iss):125.

36.

Yao Y. , Lamkin MS , Oppenheim FG (1999). Pellicle precursor proteins: acidic proline-rich proteins, statherin and histatins and their crosslinking reaction by oral transglutaminase. J Dent Res 78:1696-1703.