Methylation of arginine (Arg/R/R-Me) and lysine (Lys/K/K-Me) are essential protein post-translational modifications (PTM) of eukaryotic proteins. While detailed studies of eukaryotic methylproteomes exist for mammals (e.g., rodents, humans) and experimentally tractable model organisms (e.g., yeast), we lack species from basal (“ancient”) eukaryotic taxa. Without these species we cannot contexualise the numerous evolutionary expansions in methylproteomes of higher eukaryotes, or distinguish evolved (“new”) interactions and components from original methylation networks. The protists Trichomonas vaginalis, Entamoeba histolytica and Giardia duodenalis are among the earliest extant representatives of Eukaryota, and are major human and veterinary parasites. These protists are valuable as evolutionary models from basal eukaryotic taxa, and in representing unique phenotypic diversity (i.e. disease-causing species).
We have demonstrated functional methylproteome networks in protist lineages which pre-date current eukaryotic model species by ~1 billion years. We used sequence-based domain-mapping and 3D-structural modelling to bioinformatically curate Class I and Class V methyltransferase (MTases) enzymes in seven protist species. This identified putative lysine-MTases, including novel annotations of MTases of eukaryote Elongation Factor 1 alpha (eEF1a) in the earliest diverging extant lineages of the Eukaryota. It also demonstrated that diplomonad species including Giardia lacked methylarginine enzymes, and had converted R-Me sites and RGG motifs in substrate orthologs. To explore these losses, we performed in vitro analyses of methylation in Giardia using immunoblotting, Amino Acid Analysis, and immunoaffinity purification of methylation-modified peptides combined with LC-MS/MS. While we readily identified methyllysine in Giardia, no methylarginine was detected. This confirmed Giardia as the first eukaryote lacking conserved methylarginine networks. In contrast, we bioinformatically inferred the enzymes and preferred substrates of methylarginine are present in other protists including Entamoeba and Trichomonas, highlighting intriguing contrasts in methyl-regulation between basal species. In order to facilitate large-scale methyl-site identifications in these protists, we have validated methyl-site confidence-filtering pipelines in R software as an alternative to incompatible orthogonal methyl-peptide identification approaches (i.e., heavy-methyl SILAC). To date, we have identified >200 methyllysine sites in Giardia using these pipelines, including sites in the histone H3 variant and eEF1a which demonstrate basal origins of these eukaryotic regulatory mechanisms. However, the majority of Giardia methyllysine proteins identified were species- or lineage-specific as compared to model eukaryotes, including gene families enriched in coiled-coil features involved in cytoskeletal regulation. Together, these protists’ methylproteomes represent both specialized adaptations for parasitic lifestyles and eukaryote-conserved mechanisms, and provide a more complete understanding of the natural history of methylation through the Eukaryota.