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Tissue transglutaminase (tTGase) catalyzes both deamidation and transamidation of peptides and proteins by using a peptidyl glutamine as primary substrate. A precise consensus sequence for the enzyme is unknown and the ratio between deamidated and transamidated (or crosslinked) reaction products is highly substrate-dependent. Due to its overlapping body distribution with tTGase and ease of manipulation with tandem mass spectrometry, we used the neuropeptide substance P as a model to investigate the associated enzymatic kinetics and reaction products. Online liquid-chromatography Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR MS) combined with electron-capture dissociation (ECD) was employed to study the tTGase-induced modifications of substance P. A particular strength of ECD for peptide-enzyme reaction product monitoring is its ability to distinguish isomeric amino acids, for example, Glu and iso-Glu, by signature product ions. Our studies show that the primary reaction observed is deamidation, with the two consecutive glutamine residues converted sequentially into glutamate: first Gln(5), and subsequently Gln(6). We then applied ECD FT-ICR MS to identify the transamidation site on an enzymatically cross-linked peptide, which turned out to correspond to Gln(5). Three populations of substance-P dimers were detected that differed by the number of deamidated Gin residues. The higher reactivity of Gln(5) over Gln(6) was further confirmed by cross-linking SP with monodansylcadaverine (MDC). Overall, our approach described herein is of a general importance for mapping both enzymatically induced post-translational protein modifications and cross-linking. Finally, in vitro Ca-signaling assays revealed that the main tTGase reaction product, the singly deamidated SP (RPKPEQFFGLM-NH2), has increased agonist potency towards its natural receptor, thus confirming the biologically relevant role of deamidation.
Aïcha Hessler-Wyser, Johann Michler, Amit Sharma, Caroline Hain, Daniele Casari, Thomas Nelis
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Thomas Rizzo, Ahmed Ben Faleh, Stephan Warnke, Priyanka Bansal, Ali H Abikhodr, Vasyl Yatsyna, Natalia Yalovenko