Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC–High-Resolution ESI–MS/MS

Abstract

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the “oral protein pellicle” is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium () via the PRIDE partner repository with the data set identifier PXD014658.

The Post-Translational Modifications of Human Salivary Peptides and Proteins Evidenced by Top-Down Platforms

“…TG2 is a Ca 2+ -dependent enzyme released by the epithelial oral cells; it is negatively modulated by GTP [ 129 ] and affected by the reversible formation of an intramolecular disulfide bridge [ 130 ]. Recently, a study of our group [ 131 ] showed that also bPRPs and the P-C peptide are potential substrates of TG2. Nonetheless, they showed a very different reactivity for monodansyl-cadaverine (used as a lone pair donor).…”

“…Nonetheless, they showed a very different reactivity for monodansyl-cadaverine (used as a lone pair donor). Mass-spectrometry analyses of the reaction products highlighted that P-C, P-H, and P-D (both Pro 32 and Ala 32 variants) peptides were active substrates of TG2; II-2 was less reactive, while P-F and P-J peptides showed negligible activity [ 131 ]. MS characterization suggested that the consensus sequence for the linking is connected more to the environment of glutamine residue than to the donor ammine.…”

“…MS characterization suggested that the consensus sequence for the linking is connected more to the environment of glutamine residue than to the donor ammine. The pivotal residues characterized for P-H, II-2, and both variants of P-D evidenced …GNPQ… as the consensus sequence recognized by TG2 on bPRP peptides [ 131 ]. This consensus sequence is not present in P-F and P-J peptides and, probably for this reason, they were poor substrates for TG2.…”

“…P-C, P-H, and P-D peptides formed cyclo-derivatives after the TG2 reaction, and only specific glutamine residues were involved in the cycle formation and reacted with specific monodansyl-cadaverines [ 131 ]. The stereospecificity of TG2 was at first recognized on statherin, which under the action of TG2 forms a cyclic derivative involving almost only Gln 37 (out of seven glutamine residues) and Lys 6 (as a unique lysine residue) in the sequence ( Figure 4 ) [ 132 ].…”

Stabilization of Salivary Biomarkers

“…Dilution dictated by the addition of stabilizing solutions especially affects the detection of proteins present in low concentrations. This issue can be overcome by adding acidic buffers, such as acetate, acetic acid, or trifluoroacetic acid (TFA) to centrifuged saliva samples ,− which cause the precipitation of the most abundant proteins in saliva, such as α-amylase, carbonic anhydrase, or mucins. Acidic buffers also inhibit protease activity and induce a decrease in sample viscosity, facilitating analyte quantification .…”