HPLC-MS characterization of cyclo-statherin Q-37, a specific cyclizatin product of human salivary statherin generated by transglutaminase 2

In the present study the analytical potential of HPLC-MS/MS was utilized for the structural characterization of a post-translational modification of statherin. Human salivary statherin (Mav 5380.0 EUR 0.3 Da) is transformed by the action of transglutaminase 2 into a cyclic derivative with an average molecular mass of 5363.0 EUR 0.3 Da. The intra-molecular bridge is generated by the loss of an ammonia molecule between the unique lone-pair donating nucleophile Lys-6 and one acceptor among the seven glutamine residues of statherin. Digestion of the cyclic derivative with chymotrypsin, proteinase K, and carboxypeptidase Y, monitored by HPLC-electrospray ionization-ion trap-mass spectrometric analysis, demonstrated that cyclization involved almost specifically Gln-37 (A95%), with the percentage of Gln-39 implicated in the cross-linking being less than 5%. The main derivative was named cyclostatherin Q-37. Guinea pig transglutaminase 2 showed high affinity for statherin in vitro (Km = 0.65 EUR 0.06 lM). Cyclo-statherin was detected in vivo by HPLC-electrospray ionization ion trap-mass spectrometry analysis of whole human saliva and it accounted for about 1% of total statherin. Detection of cyclo-statherin in whole saliva is suggestive of a putative role of this molecule in the formation of the "oral protein pellicle".