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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.

SUBMITTER: Kuhn P

PROVIDER: S-EPMC2774437 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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

Kuhn Peter P Xu Qingge Q Cline Erika E Zhang Di D Ge Ying Y Xu Wei W

Protein science : a publication of the Protein Society 20090601 6

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. Un ...[more]

PMID: 19472346

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Project description:IntroductionThe human blood fluke parasite Schistosoma mansoni relies on diverse mechanisms to adapt to its diverse environments and hosts. Epigenetic mechanisms play a central role in gene expression regulation, culminating in such adaptations. Protein arginine methyltransferases (PRMTs) promote posttranslational modifications, modulating the function of histones and non-histone targets. The coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) is one of the S. mansoni proteins with the PRMT core domain.MethodsWe carried out in silico analyses to verify the expression of SmPRMTs in public datasets from different infection stages, single-sex versus mixed-worms, and cell types. The SmCARM1 function was evaluated by RNA interference. Gene expression levels were assessed, and phenotypic alterations were analyzed in vitro, in vivo, and ex vivo.ResultsThe scRNAseq data showed that SmPRMTs expression is not enriched in any cell cluster in adult worms or schistosomula, except for Smcarm1 expression which is enriched in clusters of ambiguous cells and Smprmt1 in NDF+ neurons and stem/germinal cells from schistosomula. Smprmt1 is also enriched in S1 and late female germ cells from adult worms. After dsRNA exposure in vitro, we observed a Smcarm1 knockdown in schistosomula and adult worms, 83 and 69%, respectively. Smcarm1-knockdown resulted in reduced oviposition and no significant changes in the schistosomula or adult worm phenotypes. In vivo analysis after murine infection with Smcarm1 knocked-down schistosomula, showed no significant change in the number of worms recovered from mice, however, a significant reduction in the number of eggs recovered was detected. The ex vivo worms presented a significant decrease in the ovary area with a lower degree of cell differentiation, vitelline glands cell disorganization, and a decrease in the testicular lobe area. The worm tegument presented a lower number of tubercles, and the ventral sucker of the parasites presented a damaged tegument and points of detachment from the parasite body.DiscussionThis work brings the first functional characterization of SmCARM1 shedding light on its roles in S. mansoni biology and its potential as a drug target. Additional studies are necessary to investigate whether the reported effects of Smcarm1 knockdown are a consequence of the SmCARM1-mediated methylation of histone tails involved in DNA packaging or other non-histone proteins.

Dataset Information

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

Publications

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

Similar Datasets

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