Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis

Abstract

The polyamines spermidine and spermine, and their precursor putrescine, have been shown to play an important role in cell migration, proliferation, and differentiation. Because of their polycationic property, polyamines are traditionally thought to be involved in DNA replication, gene expression, and protein translation. However, polyamines can also be covalently conjugated to proteins by transglutaminase 2 (TG2). This modification leads to an increase in positive charge in the polyamine-incorporated region which significantly alters the structure of proteins. It is anticipated that protein polyamine conjugation may affect the protein-protein interaction, protein localization, and protein function of the TG2 substrates. In order to investigate the roles of polyamine modification, we synthesized a spermine-conjugated antigen and generated an antiserum against spermine. In vitro TG2-catalyzed spermine incorporation assays were carried out to show that actin, tubulins, heat shock protein 70 and five types of histone proteins were modified with spermine, and modification sites were also identified by liquid chromatography and linear ion trap-orbitrap hybrid mass spectrometry. Subsequent mass spectrometry-based shotgun proteomic analysis also identified 254 polyaminated sites in 233 proteins from the HeLa cell lysate catalyzed by human TG2 with spermine, thus allowing, for the first time, a global appraisal of site-specific protein polyamination. Global analysis of mouse tissues showed that this modification really exists in vivo. Importantly, we have demonstrated that there is a new histone modification, polyamination, in cells. However, the functional significance of histone polyamination demands further investigations.

β 2 -adrenoceptor-induced modulation of transglutaminase 2 transamidase activity in cardiomyoblasts

“…spermine, spermidine, and putrescine can be covalently attached onto proteins via TG2-mediated transamidation activity, resulting in the incorporation of a positively charged group into the target protein. Thus, TG2-mediated polyamination may promote changes in protein conformation, which could lead to alterations in protein function (Yu et al, 2015). For example, the TG2-mediated incorporation of polyamines into RhoA results in constitutive G-protein activity (Makitie et al, 2009;Shin et al, 2008;Singh et al, 2001), whereas the incorporation of polyamines into phospholipase A2 results in a 2-3 fold increase in enzymic activity (Cordella-Miele et al, 1993).…”

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

“…Proposed novel TG2 targets not appearing in the TG2 substrate database [71] or identified by Yu et al [72] …”

Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps

“…22, 29, 30, 39, 40, 41, 42 Cornification and crystallin modifications are particularly relevant to this study as in both processes TGase-mediated formation of bis( γ -glutamyl)polyamine cross-links between proteins by TGase 1 and 2, respectively, was observed 29, 30, 40 in parallel to protein cross-linking through ɛ ( γ -glutamyl)lysine bonds. NET protein fractions contain both bis( γ -glutamyl)polyamine and ɛ ( γ -glutamyl)lysine bonds suggesting that TGase-mediated cross-linking contributes to selective enrichment of specific proteins in the NET and its stabilization.…”