Pellicle precursor protein crosslinking: Characterization of an adduct between acidic proline-rich protein (PRP-1) and statherin generated by transglutaminase

Recent work with oral transglutaminase indicated that this enzyme, derived from oral epithelial cells, crosslinked pellicle precursor proteins which m ay be important in the formation of the acquired enamel pellicle. The purpo se 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 t ransglutaminase-catalyzed reaction between glutaminyl and lysyl side-chains , leading to a covalent bond formation. Enzymatic reaction products were an alyzed by SDS-PAGE and reverse-phase HPLC. The SDS electrophoretogram revea led a protein band with an apparent molecular weight of 32 kDa, which is co nsistent with the combined apparent molecular weight of acidic PRP-1 (24 kD a) and statherin (8 kDa). A reaction product isolated by HPLC was character ized by amino acid analysis, which showed a stoichiometry consistent with b eing 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 ad duct was not detected when the lysines of both substrates were modified by reductive methylation prior to the enzymatic reaction. In addition, amino a cid analysis and mass spectrometry confirmed the presence of a gamma-glutam yl-epsilon-lysine dipeptide after enzymatic hydrolysis and the absence of t his dipeptide after acid hydrolysis. Analysis of the data obtained indicate s that oral transglutaminase is capable of crosslinking acidic PRP-1 and st atherin in vitro. In addition, this finding exemplifies the potential of po st-secretory processing of salivary proteins, which may represent an additi onal mechanism to generate new protein species.