Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages

Plasmodium falciparum culture

Plasmodium falciparum 3D7 was cultured in complete RPMI (1640 (Invitrogen Corporation, USA), 50 mg/L hypoxanthine (Sigma Aldrich Co., USA), 0.5 g/L Albumax I (Gibco, Thermofisher Scientific Inc., USA) and 2 g/L sodium bicarbonate (Sigma Aldrich Co., USA) using O⁺ human erythrocytes (4% haematocrit) under mixed gas (5% O₂, 5% CO₂ and 90% N₂). Cultures were synchronized with 5% sorbitol for at least two successive cycles and harvested using saponin treatment.

Immunofluorescence assay

Thin smears were prepared from Plasmodium falciparum culture at ring, trophozoite and schizont stages. The glass slides were air dried and fixed with pre-chilled absolute methanol for 30 minutes. The smears were incubated with 4% BSA in PBS for 2 h at room temperature (RT) to block the non-specific binding. After washing with PBS, the smears were probed with anti-mono/dimethyl lysine polyclonal antibody (abcam; ab23366)(1:20) for overnight at 4 °C, followed by Alexa-Fluor 488 conjugated goat anti-rabbit IgG antibody (A11008; Thermofisher Scientific Inc. USA) (1:200) for 1 h at RT. The slides were washed and mounted with 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI, Molecular Probes, USA) antifade solution (Molecular Probes, USA). Images were captured using a Nikon A1-R confocal microscope.

Preparation of parasite lysate, Immunoprecipitation and Western blot analysis

Immuno-precipitation was performed using Pierce® Crosslink Immunoprecipitation Kit (Thermofisher Scientific Inc., USA) according to manufacturer’s protocol. Briefly, parasite cell lysates from synchronized cultures of three asexual stages (ring, trophozoite and schizont) were prepared using IP lysis buffer. The stage specific cell lysates were incubated overnight at 4 °C with anti-mono/dimethyl (abcam; ab23366) or anti-trimethyl lysine (Immunechem Pharmaceutical Inc.; ICP0601) antibodies, cross-linked with Protein A/G PlusAgarose by DSS cross-linker. The antibody cross-linked resin was washed with TBS followed by two washes with lysis buffer. Finally, the resin was washed with conditioning buffer and antibody bound proteins were eluted with elution buffer.

Also, the peptides obtained after trypsin digestion of trophozoite stage parasite lysates were lyophilized, desalted and incubated overnight at 4 °C with anti-mono/dimethyl and anti-trimethyl lysine antibodies cross-linked with Protein A/G Plus agarose. The eluted peptides were lyophilized and separated into 12 fractions using hydrophilic liquid interaction chromatography (HILIC) over one hour. Each fraction was separately analyzed on LC-MS/MS.

For western blot analysis, equal amount (50 μg) of total protein from three stages were resolved on 10% SDS-PAGE and transferred onto PVDF membrane (Merck Millipore, Merck KGaA, USA) pre-activated with methanol. The membrane was blocked with 4% BSA and incubated with polyclonal anti-methyllysine antibodies (ImmuneChem Pharmaceuticals Inc, Canada, ICP0601; 1:50) overnight at 4 °C. After three washings with PBST, the membrane was incubated with HRP conjugated anti-rabbit IgG secondary antibody (Sigma Aldrich Pvt. Ltd., USA) for 1 h at RT. The membrane was developed and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermofisher Scientific Incorporation, USA) as per manufacturer’s instructions.

For confirmation of proteins in the IP experiments, a fraction of IP eluates pulled using anti-methyllysine antibodies were boiled in 4X sample loading buffer, separated on 10% SDS-PAGE gels and transferred to a nitro-cellulose membrane. The membrane was blocked with blocking buffer (ODYSSEY infrared imaging systems; LI-COR) and probed with protein specific antisera (anti-PfP12; a 6-cysteine protein and anti-PfTSN; Tudor Staphylococcal Nuclease) using anti-mice IRDye 800CW secondary antibodies (LI-COR). Protein bands were imaged in ODYSSEY Infrared Imager (LI-COR).

Tryptic digestion and LC-MS/MS analysis

Eluted proteins from immunoprecipitation were subjected to in-solution digestion. Samples were subjected to subsequent reduction and alkylation of disulfide bonds with 10 mM dithiothreitol (DTT) and 40 mM iodoacetamide for 1 hr. at room temperature. Digestion was performed using trypsin (1:50, enzyme: total protein) (Promega corporation, USA) for overnight at 37 °C and stopped by adding 0.1% trifluoroacetic acid. The samples were cleared by centrifugation at 10000 rpm for 10 min. The digested proteins were concentrated using Speed Vac (Thermofisher Scientific Inc., USA) and analyzed on Orbitrap Velos Pro mass spectrometer coupled with nano-LC 1000 (Thermofisher Scientific Inc., USA). The peptide mixtures were loaded on to a reverse phase C-18 pre-column (Acclaim PepMap, 75 μm × 2 cm, 3 μm, 100 A°, Thermofisher Scientific Inc., USA), in line with an analytical column (Acclaim PepMap, 50 μm × 15 cm, 2 μm, 100 A°). The peptides were separated using a gradient of 5% to 50% of solvent B (0.1% formic acid in 95/5 acetonitrile/water) in 180 min for immunoprecipitates and 120 min for HILIC fractions. The peptides were analyzed in data dependent mode where the precursors were acquired (MS) in Orbitrap at a resolution of 60000 and a minimum of 1000 counts were needed to trigger the MS/MS. Top 20 precursors were allowed to fragment using CID (collision induced dissociation) in Ion trap with collision energy of 35 for the immunoprecipitates. For HILIC fractions, precursors were fragmented using high-energy collision dissociation (HCD) and detected in Orbitrap at a resolution of 7500. Charge state screening of precursors and monoisotopic precursor selection was enabled. Unassigned and singly charged ions were rejected. The parent ions once fragmented were excluded for next 50 s with exclusion mass width of ±10 ppm. The lock mass (m/z 445.120024) enabled the accurate measurement in MS for immunoprecipitated samples and in both MS as well as MS/MS for HILIC fractions. The acquired spectra were analyzed using SEQUEST algorithm in Proteome Discoverer (PD; version 1.4) software, with a precursor tolerance of 20 ppm and tolerance of 0.6 Da for MS/MS for CID and 0.1 Da for HCD against P. falciparum database version 10 downloaded from PlasmoDB²⁷. Carbamidomethyl (C), Deamidation (NQ) and mono-methyl, di-methyl and trimethyl (K) were set as variable modifications and five missed cleavages were allowed. The resultant identified peptides were validated using Percolator at 5% False Discovery Rate (FDR) (q value < 0.05), which uses PEP (Posterior Error Probability) and q value for validations.

Lysine methylation motif analysis

The motif search included six amino acid residues N-terminal and C-terminal to each methylation sites. Putative lysine methylation sites were analyzed, based on previously known methyl lysine associated motifs in other organisms, for example LK and MK. To determine the sequence motifs, we developed a PERL script to fetch six amino acids upstream as well as downstream of a central lysine. The frequency of amino acids near lysine residue was also analyzed using WebLogo²⁸. To compare neighboring residues in the methylated and non methylated lysines in the proteomics identified proteins, we used Two Sample Logo²⁹.

Gene Ontology (GO) analysis

GO enrichment was performed for functional classification and cellular localization of the methylated proteins using PlasmoDB (version 13.0).

Interaction analysis using STRING

We downloaded the STRING³⁰ Protein-Protein Interaction (PPI) network for P. falciparum and converted it into R object using igraph package³¹. Next, we removed the Edges with no experimental evidence of interaction (Experimental < 0). However, the network contained redundant edges and loops which were removed too, using simplify() function from igraph package. SET domain containing proteins along with its top 10 nearest neighbors were retrieved and the network was visualized using Cytoscape³².

Anti-methyl lysine specific antibodies recognize Plasmodium proteins at asexual blood stages

Plasmodium genome sequencing analysis has revealed existence of a number of methyltransferases, particularly SET-domain-containing proteins and demethylases that are responsible for lysine methylation in the genome²²,³³. To assess the extent of lysine methylation in Plasmodium proteins in asexual blood stages, we tested the reactivity of commercially available anti-mono/di methyl lysine and anti-trimethyl lysine antibodies with the parasite lysate and with intact blood stage P. falciparum parasites by western blot and immunofluorescence analysis respectively. As shown in Fig. 1A, anti-methyl lysine specific antibodies recognized specific bands at ring, trophozoite and schizont stages, although the extent of reactivity appeared more at the schizont stage. The results were further confirmed by immunofluorescence assay where a considerable staining was observed at all the three parasite blood stages, thereby indicating the extensive methylation of Plasmodium proteins at lysine residues. Staining was mainly observed in the periphery of nucleus as blue DAPI stain overlapped with the anti-lysine antibody immunostains, however some staining was also seen in parasite cytoplasm (Fig. 1B).

Identification of P. falciparum lysine methylated proteins

Next, we carried-out global proteome analysis of lysine methylated Plasmodium proteins at asexual blood stages by immunoprecipitating the proteins from Plasmodium lysates prepared from the three asexual blood stages; ring, trophozoite and schizont using either anti-mono/di methyl lysine and anti-trimethyl lysine antibodies. Mass spectroscopic analysis of immunoprecipitated proteins identified a total of 570 putative lysine methylated proteins in P. falciparum asexual blood stages (Fig. 2). The two antibodies pulled-down approximately the same number of proteins at each stage, with considerable overlap. The one hundred and thirty two proteins identified in the proteome analysis were common to all the three stages (Fig. 2, Supplementary Table 1). To determine whether these putative methylated proteins have methylated lysine residues, we analyzed the spectra of the peptides generated in mass spectrometric analysis. As shown in Table 1, we could identify 364 K-methylated sites on 266 peptides corresponding to Plasmodium proteins. The representative spectra of few of the methylated peptides corresponding to Plasmodium proteins are shown in Supplementary Fig. 1.

A number of reports have shown that the pre-fractionation of tryptic peptides either by SCX or HILIC provides a sort of enrichment of methylated peptides that can be better captured on LC-MS/MS for site identification. Between the two methods, HILIC has provided more number of methylation sites as compared to SCX in S. cerevasiae³⁴. We performed HILIC chromatography analysis on trypsin digested parasite lysate generated from trophozoite stage. Twelve fractions were collected in two replicates and each of the fractions was separately analyzed on LC-MS/MS. As shown in Table 1, we could identify 247 sites in 236 peptides corresponding to Plasmodium proteins. Representative spectra of few of these K-methylated peptides are shown in the Supplementary Fig. 2. In total, 605 methylated lysine sites in 502 peptides were identified corresponding to 422 Plasmodium proteins. A couple of previous reports in human/mouse cell lines and Saccharomyces cerevisiae have shown that many of the lysine methylated sites are associated with EK, LK and MK motifs. We searched the motifs in 570 putative lysine methylated proteins identified by immunoprecipitation of parasite lysates. We could observe LK and EK motifs in many Plasmodium proteins (Fig. 3). The “Two Sample Logo” visualization of the residues surrounding methylated and non methylated lysines amongst the proteomics identified proteins reveal that the residues surrounding methylated lysines are different from those surrounding the non methylated lysines (Supplementary Fig. 4). Among the Plasmodium methylated peptides, we identified 152 monomethyl, 249 dimethylated and 210 trimethylated sites in the mass spectrometry analysis corresponding to these proteins (Fig. 3).

To confirm the identity of non-histone lysine methylated proteins, we subjected the immunoprecipitated Plasmodium lysate to western blot analysis using anti-PfP12 or anti-PfTSN antisera. As shown in Fig. 4, PfP12 and PfTSN are detected in IP lysate, thus confirming the lysine methylation of the proteins. Altogether, several mass spectrometric analysis runs as well as western blot analysis indicate extensive lysine methylation of Plasmodium proteins at asexual blood stages.

Classification of lysine methylated proteins

Mass spectrometry analysis of the anti-methyl lysine antibody immuno-precipitated proteins from the Plasmodium lysates suggested that over 10% of the proteins encoded in the P. falciparum genome are modified by lysine methylation. To get insight into the role of lysine methylation in the parasite growth and development, we utilized the fully annotated genome database (PlasmoDB) to describe correlations for lysine methylated proteins, their families, subcellular localization and biological function. The identified lysine methylated proteins were classified based on their subcellular localization with GO term analysis using PlasmoDB. As seen in Fig. 5A, we were able to obtain the GO assigned localization of only 78% of the lysine methylated proteins, since a large number of the query proteins were hypothetical. The percentage of proteins with known or predicted subcellular localization was highest for cytoplasmic proteins (Fig. 5A). Many of the identified lysine methylated proteins appeared to be part of protein-protein or protein-DNA complexes associated with chromosome or ribosome.

The diverse localization profile of lysine-methylated proteins suggests that this modification regulates a wide range of cellular functions. To define the functional classes of lysine methylated proteins, we data mined the P. falciparum annotated genome database for all identified methylated proteins. Similar to the localization GO terms, we were able to predict functional classes for only 62% of the identified lysine methylated proteins (Fig. 5B). Many of these annotated proteins seem to be associated with the transport/trafficking processes, chromosomal organization and translation regulation (Fig. 5B).

To get insights into the role(s) of lysine methylated Plasmodium proteins identified by mass spectrometry analysis, we sorted the proteins based on other conserved domains. As shown in Fig. 5C, several of these proteins belong to major super families such as protein kinase C (PKc), ATS, P-loop_ NTPase, PHD_SF and AdoMet_MTases_SF. The set also included 21 PfEMPs and 17 rifin proteins, indicating a role of lysine methylation in antigenic diversity in P. falciparum.

Lysine methyltransferase-substrate interactome networks and cross talks between PTMs

For further exploration of the breadth of Plasmodium protein lysine methylation and associated lysine methyltransferases (KMTs), we generated KMT-substrate networks using STRING database and published literature. These networks are depicted in Supplementary Fig. 3 and are described in Supplementary Table 2. All the Plasmodium SET domain proteins show a number of associated partners and many of them are non-histone proteins such as endoplasmin putative (GRP94), bromodomain protein 1 (BP1), proliferating cell nuclear antigen1, guanylyl cyclase (GCalpha). Importantly, some of the lysine-methylated proteins identified in our proteome analysis are also part of this KMTs-substrate network (Supplementary Table 2). For example endoplasmin putative (GRP94) and proliferating cell nuclear antigen 1 (PCNA1) proteins are identified in STRING as well as in our mass spectrometry analysis. We also observed associations among the set domain proteins. For example SET3 protein shows association with SET8 and SET4 proteins in the STRING analysis (Supplementary Table 2).

Extensive cross talk has been reported and predicted between different PTMs in different organisms, including Plasmodium. We compared the methylated lysine containing proteins with the previously published studies for phosphorylation and acetylation in P. falciparum⁵,⁶,⁸,³⁵. This comparison showed that 209 lysine-methylated proteins were phosphorylated too. Further, 173 Plasmodium proteins were acetylated as well as lysine methylated and 113 proteins possess all three modifications (Supplementary Table 3). Several proteins from the STRING analysis were either acetylated or phosphorylated (Supplementary Table 2), thus indicating extensive cross talks between various PTMs.

Supplementary Information

Supplementary Information

Supplementary Information

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Supplementary Materials