Delineating Anopheles gambiae coactivator associated arginine methyltransferase 1 automethylation using top–down high resolution tandem mass spectrometry

Abstract

Coactivator-associated arginine methyltransferase 1 (CARM1), originally defined as a coactivator for steroid receptors, is a member of the protein arginine methyltransferases. Here, we report the discovery and characterization of an automethylation event by AgCARM1, a CARM1 homologue in the mosquito Anopheles gambiae, using top-down high resolution tandem mass spectrometry, which allows fine mapping of modifications in the intact protein accurately and quantitatively without priori knowledge. Unexpectedly, we found that AgCARM1 has already been predominantly dimethylated during its expression in Escherichia coli. A single arginine methylation site, R485, was identified which is conserved among CARM1 in insects. No methylation was observed in the intact AgCARM1 R485K mutant where R485 is mutated to lysine, which confirms that R485 is the only detectable methylation site. Using AgCARM1 methyltransferase defective mutants, we confirmed that this is an automethylation event and show the automethylation of AgCARM1 occurs intermolecularly. This study represents the first comprehensive characterization of an automethylation event by top-down mass spectrometry. The unexpected high percentage of automethylated recombinant AgCARM1 expressed in E. coli may shed light on other bacterially expressed post-translational modifying enzymes, which could be modified but overlooked in biochemical and structural studies. Top-down high resolution tandem mass spectrometry thus provides unique opportunities for revealing unexpected protein modification, localizing specific modification to one amino acid, and delineating molecular mechanism of an enzyme.

O-GlcNAcylation of co-activator-associated arginine methyltransferase 1 regulates its protein substrate specificity

“…The high-resolution FT-ICR (Fourier transform-ion cyclotron resonance) MS analysis of Halo-tag-purified CARM1 was performed as described previously [26].…”

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

“…The top-down MS strategy allows analysis of intact proteins to obtain a comprehensive assessment and localization of post-translational modifications (PTMs) and point mutations with full sequence coverage and without a priori knowledge. [27][28][29][30][31][32][33][34][35][36][37] It starts with measuring molecular weights of intact proteins followed by fragmentation of the protein in the mass spectrometer with tandem mass spectrometry (MS/MS) such as electron capture dissociation (ECD) 38 to obtain sequence information. ECD has been demonstrated to be especially valuable for mapping phosphorylation sites, because it preserves the labile PTMs during MS/MS process.…”

The enzymatic activity of Arabidopsis protein arginine methyltransferase 10 is essential for flowering time regulation

“…So far, four mammalian PRMTs, PRMT1, CARM1/PRMT4, PRMT6 and PRMT8, were found to have auto-methylation activity, and the auto-methylated arginines were identified by mass spectrometry (Frankel et al, 2002;Sayegh et al, 2007;Kuhn et al, 2009Kuhn et al, , 2011Gui et al, 2011). For functional dissection, CARM1 with a mutation in the auto-methylation site was found to have comparable enzymatic activity to the wild-type CARM1 (Kuhn et al, 2011).…”

“…PRMTs methylate a wide variety of histone and nonhistone substrates, and some PRMTs including PRMT1, CARM1/PRMT4, PRMT6 and PRMT8 also have auto-methylation activities (Frankel et al, 2002;Sayegh et al, 2007;Kuhn et al, 2009;Gui et al, 2011;Kuhn et al, 2011). The automethylation sites of CARM1s in different species were mapped by mass spectrometry.…”

“…The automethylation sites of CARM1s in different species were mapped by mass spectrometry. Amino-acid substitutions in these auto-methylation sites seem to have no effect on CARM1 enzymatic activity (Kuhn et al, 2009(Kuhn et al, , 2011. Nevertheless, the loss of auto-methylation was reported to impair the role of CARM1 in transcriptional activation and alternative splicing, which indicates the importance of auto-methylation in PRMT's biological function (Kuhn et al, 2011).…”