Deamidation and Transamidation of Substance P by Tissue Transglutaminase Revealed by Electron‐Capture Dissociation Fourier Transform Mass Spectrometry

Abstract

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 cross-linked) 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 Gln 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-NH(2) ), has increased agonist potency towards its natural receptor, thus confirming the biologically relevant role of deamidation.

Tissue Transglutaminase-mediated Glutamine Deamidation of β-Amyloid Peptide Increases Peptide Solubility, Whereas Enzymatic Cross-linking and Peptide Fragmentation May Serve as Molecular Triggers for Rapid Peptide Aggregation

“…More importantly, we observed that conversion from Gln 15 to Glu 15 now provides an additional cleavage site for Glu-C endoprotease proteolysis at residue Glu 15 , resulting in an additional proteolytic fragment of residues A␤ 16 -22 as compared with control A␤ [12][13][14][15][16][17][18][19][20][21][22] (Gln 15 ) digestions (data not shown). Similarly, we found that in substance P, Gln 5 is readily deamidated by TGase, whereas Gln 6 appeared to be a less favorable substrate, and glutamine deamidation to glutamate also introduces a new cleavage site for Glu-C proteolysis (29).…”

“…Nevertheless, we were able to induce stable monodansylcadaverine incorporation in the A␤ peptide, through a transamidation reaction, using intact CHO cells (data not shown). Monodansylcadaverine cross-linking with glutamine residues in Substance P in vitro has been recently been reported using the electron capture dissociation analytical approach (29).…”

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

“…ECD has traditionally been achieved on Fourier-transform ion cyclotron resonance mass spectrometers (92,93). In a recent report, however, ECD fragmentation of sialylated N-linked glycopeptides was performed on a radio-frequency quadrupole ion trap (47).…”

Cellular Functions of Tissue Transglutaminase

“…This TG2-induced deamidation of crystallins is thought to contribute to age-dependent lens opacification in humans. Moreover, deamidation of Gln15 in β-amyloid peptide was shown to reduce its solubility (Schmid et al, 2011), whereas deamidation of Gln6 in substance P increased agonist potency toward its receptor (Fornelli et al, 2011), thus expanding the potential pathophysiological role of these TG2-dependent reactions (Fig. 1.3(3)).…”