ABSTRACT: Due to the distinctive features of oral mucosa, determination of the proteins involved in the formation of “oral protein pellicle” is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides (bPRPs) named P-H, P-D, P-F, P-J, and II-2 as substrates of transglutaminase-2. The reactivity of P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated at diverse temperatures with two different transglutaminases (type 2; guinea pig and human), in the presence or in the absence of monodansyl-cadaverine. High resolution HPLC-ESI-MS and MS/MS analyses of the reaction products highlighted that P-H, and P-D were active substrates of transglutaminase-2, II-2 was less reactive and the reactivity of P-F and P-J was negligible. All the peptides formed cyclo-derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine. Q29 for P-H, Q37 for P-D, and Q21 for II-2 were the principal reactive residues. One or two secondary glutamine residues were hierarchically susceptible to reaction with dansyl-cadaverine. The main consensus sequence recognized by transglutaminase-2 in basic proline-rich peptides was GNPQ, and, despite the great similarity of P-F and P-J with the other salivary peptides, their reactivity was negligible, probably because the GNPQ consensus sequence was missing in their sequence. The reactivity of P-D A32 variant was lower than that of P-D P32 peptide suggesting that the more rigid peptide structure was a better substrates for transglutaminase-2. The reactivity of P-C was high, but followed rules different from the other basic peptides under study, Q41 being the principal reactive residue, confirming that P-C is not strictly related to the basic proline-rich proteins. The reactivity of statherin was very high too, and the manual inspection of the MS/MS fragmentation patterns of the reaction products confirmed, as determined in previous studies, that Q37 is the pivotal residue, Q39 and Q40 the secondary residues recognized by transglutaminase-2. Moreover, Q39 and Q40 residues were more reactive in cyclo-statherin Q37 than in statherin, suggesting that cycle formation increases the reactivity of the secondary glutamine residues. Finally, the physiological temperature (37°C) was the best for the reaction and no significant qualitative differences were observed using human or guinea pig transglutaminase-2.
Tiziana Cabras 
