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

Dept. of Periodontology and Oral Biology, Boston University School of Dental Medicine, 700 Albany Street, Boston, MA 02118, USA

M.S. Lamkin

Dept. of Periodontology and Oral Biology, Boston University School of Dental Medicine, 700 Albany Street, Boston, MA 02118, USA

FG Oppenheim

Dept. of Periodontology and Oral Biology, Boston University School of Dental Medicine, 700 Albany Street, Boston, MA 02118, USA, Dept. of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, USA

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 -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.

Key Words: acquired enamel pellicle • transglutaminase • salivary proteins.

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Journal of Dental Research, Vol. 79, No. 4, 930-938 (2000)
DOI: 10.1177/00220345000790040801


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