Proteomic Identification and Analysis of Arginine-Methylated Proteins

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Proteomic identification and analysis of arginine-methylated proteins of Plasmodium falciparum at asexual blood stages Mohammad Zeeshan, Inderjeet Kaur, Joseph Joy, Ekta Saini, Gourab Paul, Abhinav Kaushik, Surbhi Dabral, Asif Mohmmed, Dinesh Gupta, and Pawan Malhotra J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b01052 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 11, 2016

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Journal of Proteome Research

Proteomic identification and analysis of argininemethylated proteins of Plasmodium falciparum at asexual blood stages Mohammad Zeeshan§1,2, Inderjeet Kaur§1, Joseph Joy2, Ekta Saini1, Gourab Paul1, Abhinav Kaushik, Surbhi Dabral1, Asif Mohmmed3, Dinesh Gupta∗2 and Pawan Malhotra∗1

1

Malaria Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi – 110067, India 2

Translational Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi-110067, India 3

Parasite Cell Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi-110067, India

Tel. +91-11-26741358; Fax: +91-11-26742316 §

Authors have contributed equally.

*To whom correspondence should be addressed. E-mail: [email protected] and [email protected]

ACS Paragon Plus Environment

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Abstract

Plasmodium falciparum undergoes a tightly regulated developmental process in human erythrocytes, and recent studies suggest an important regulatory role of post-translational modifications (PTMs). As compared to Plasmodium phosphoproteome, little is known about other PTMs in the parasite. In the present study, we performed a global analysis of asexual blood stages of Plasmodium falciparum to identify arginine-methylated proteins. Using two different methyl arginine-specific antibodies we immunoprecipitated the arginine-methylated proteins from the stage-specific parasite lysates and identified 843 putative arginine methylated proteins by LC-MS/MS. Motif analysis of the protein sequences unveiled that the methylation sites are associated with the previously known methylation motifs such as GRx/RGx, RxG, GxxR or WxxxR. We identified Plasmodium homologs of known arginine-methylated proteins in trypanosomes, yeast and human. Hydrophilic interaction liquid chromatography (HILIC) was performed on the immunoprecipitates from the trophozoite stage to enrich arginine-methylated peptides. Mass spectrometry analysis of immunoprecipitated and HILIC fractions identified 55 arginine methylated peptides having 62 methylated -arginine sites. Functional classification revealed that the arginine-methylated proteins involved in RNA metabolism, protein synthesis, intracellular protein trafficking, proteolysis, protein folding, chromatin organization, hemoglobin metabolic process and several other functions. Summarily, these findings suggest that protein methylation of arginine residues is a widespread phenomenon in Plasmodium, and the PTM may play an important regulatory role in a diverse set of biological pathways, including host-pathogen interactions.

Keywords: Plasmodium, parasite, arginine methylation, LC-MS/MS, Malaria


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Introduction Malaria remains an important human disease with about two hundred million infections worldwide, annually 1. Among the five species of Plasmodium that infect humans, P. falciparum is mainly responsible for fatalities. The complex life cycle of the malaria parasite involves mosquito and human hosts; however the blood stage of the parasite accounts for the clinical symptoms in humans. The P. falciparum intraerythrocytic development is a tightly regulated process, however various common developmental gene regulatory processes are yet to be discovered in the parasite. Similar to other eukaryotes, one of the mechanisms involved in developmental regulation of Plasmodium is the Post-Translational Modifications (PTMs) of proteins, which influences many biological processes. In malaria parasites, PTMs such as protein phosphorylation, ubiquitination, acetylation and palmitoylation have been described, however only protein phosphorylation and dephosphorylation processes have been studied rigorously 1-8. In the last decade, methylation of proteins has been shown to be of common occurrence in human, Saccharomyces cerevisiae and trypanosomes 9-12. The SwissProt database has ranked protein methylation as the fourth most frequent modification 13. Protein methylation is mainly found on lysine and arginine residues, although there are reports of methylation on histidine and glutamic acid too 14. Methylation, especially lysine methylation, is well studied in histones and it involves addition of one to three methyl groups on the amino acid’s-amine group to form mono, di or tri-methyl-lysine residues. Lysine methylation in conjunction with acetylation and phosphorylation controls protein-protein interactions and affects chromatin transcription 1, 15-17. Recent research has shown arginine methylation as a common posttranslational modification in eukaryotes with important role in transcription, translation, nucleo-cytoplasmic transport, signaling, DNA repair, RNA processing and splicing 18-21. Methylation of arginine involves addition of methyl groups to the amino acid guanidino


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group, brought about by protein arginine methyltransferases (PRMTs). The PRMTs are unique to eukaryotes and have been classified into four types, designated I to IV, based on the number of methyl groups added and the position of the modified guanidine group 22-24. The PRMTs catalyze the transfer of methyl group from S-adenosyl methionine (AdoMet) to arginine residues in proteins. A number of proteome-wide studies have been carried-out to identify arginine or lysine– methylated proteins in human, S. cerevisiae and Trypanosoma 9, 11, 25-26. Arginine methylated proteins in these organisms were isolated and identified either by using anti-methyl arginine antibodies specific against RG-rich sequences or by stable isotope labeling of amino acids in cell culture (SILAC) using [13CD3 ] S-adenosyl methionine. In P. falciparum, three PRMTs have been identified using in silico analysis 27. In the present study, we performed an in-depth proteome analysis of arginine-methylated proteins isolated from three asexual blood stages of P. falciparum using anti-methyl arginine antibodies and identified 168 arginine methylated Plasmodium proteins by MS/MS and homology analysis. Some of the identified proteins appear to be involved in known methyl arginine-associated functions. These results suggest an important role of protein arginine methylation in various regulatory processes of the parasite. Experimental Procedures Plasmodium falciparum culture: Plasmodium falciparum 3D7 was cultured in complete RPMI (RPMI 1640 (Invitrogen, USA), 50mg/L hypoxanthine (Sigma, USA), 0.5 g/L Albumax I (Gibco, USA) and 2 g/L sodium bicarbonate (Sigma, USA) using 4% haematocrit under mixed gas (5% O2, 5% CO2 and 90% N2). The cultures were synchronized with 5% sorbitol for at least two successive cycles. Immunofluorescence assay: Plasmodium falciparum culture at the ring, trophozoite and schizont stages were smeared on glass slides, dried and fixed with pre-chilled absolute


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methanol for 30 min. Nonspecific binding sites were blocked with 4% BSA in PBS for 2 h at room temperature (RT) and probed with anti-MMA rabbit monoclonal antibody (1:20) for overnight at 4° C, followed by Alexa-Fluor 488 conjugated goat anti-rabbit IgG antibody (1:200) for 1 h at RT. The slides were washed, mounted with 4′, 6-diamidino-2-phenylindole dihydrochloride-antifade solution (Molecular Probes, USA) and analyzed using a Nikon A1R confocal microscope. Immunoblot analysis: Immunoblot analysis was carried-out with parasite lysates at three asexual blood stages. Briefly, parasites were released from infected erythrocytes by 0.1% (w/v) saponin treatment. These parasites were lysed and proteins in the parasite lysates were resolved on 10% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose membrane. Immunoblotting was performed using anti-MMA and anti-DMA antibodies (1:1000 dilution), followed by incubation with horseradish peroxidase-labeled anti-mouse IgG. Reaction was developed with 3,3’-dianitrobenzidine as a substrate. Preparation of parasite lysate and immunoprecipitation: Synchronized parasite cultures were harvested by saponin treatment at different stages of parasite life cycle (ring, trophozoite and schizont stage), and cell lysates were prepared using IP lysis buffer. Immunoprecipitation Immunoprecipitation

was Kit

performed (Thermofisher

in

duplicate,

Scientific

Inc.,

using USA)

Pierce® according

Crosslink to

the

manufacturer’s protocol. Briefly, stage-specific cell lysates (1-2 mg of total protein) were incubated overnight at 4º C with anti-Monomethyl arginine antibody (anti-MMA) and antiDimethyl arginine antibody (anti-DMA) (Immunechem) cross-linked with Protein A/G Plus Agarose by disuccinimidyl suberate (DSS) cross-linker. The resin was washed with Tris Buffer Saline (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.


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Also, trophozoite stage parasites (duplicate) were lysed and digested with trypsin, followed by Hydrophilic Liquid Interaction Chromatography (HILIC). The peptides were lyophilized, desalted and incubated overnight at 4º C with anti-MMA and anti-DMA antibodies crosslinked with Protein A/G Plus Agarose. The eluted peptides were lyophilized and resuspended in buffer B (0.1% formic acid, 95% acetonitrile). The peptides were loaded onto a HILIC column (Agilent) and separated into 12 fractions with a shallow gradient of buffer A (0.1% formic acid) over one hour. Each fraction was separately analyzed on LC-MS/MS. Tryptic digestion and LC-MS/MS analysis: Eluted proteins were subjected to in-solution digestion. Samples were subjected to subsequent reduction and alkylation of disulfide bonds with 10 mM Dithiothreitol (DTT) and iodoacetamide (40 mM) for 1 hr., at room temperature. Trypsin was added to the samples in a ratio of 1:50 (enzyme:total protein) and incubated overnight at 37° C. Digestion reaction was stopped by adding 0.1% trifluoroacetic acid and samples were cleared by centrifuging at 10000 rpm for 10 min. The digested protein samples were concentrated using Speed Vac concentrator (Thermofisher Scientific Inc., USA) and analyzed on Orbitrap Velos Pro mass spectrometer coupled with nano-LC 1000 (Thermofisher Scientific Inc., USA). Tryptic digested peptide mixtures were loaded on to a reverse phase C-18 pre-column (Acclaim PepMap, 75 µm x 2 cm, 3µm, 100A°, Thermofisher Scientific Incorporation, USA), which was in line with an analytical column (Acclaim PepMap, 50µm x 15 cm, 2µm, 100A°). 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 first acquired (MS) in Orbitrap at a resolution of 60000. The peptides from immunoprecipitated samples were analyzed in data dependent mode and top 20 precursors were allowed to fragment using CID (collision induced dissociation) in Ion trap with collision energy of 35. For HILIC fractions, top 20 precursors were fragmented using high-energy collision dissociation (HCD) of 40 and


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detected in Orbitrap at a resolution of 7500. The mass window for precursor ion selection was 1.2 Da and a minimum of 1000 counts were needed to trigger the MS/MS. Charge state screening of precursors and monoisotopic precursor selection was enabled. Unassigned charge state and singly charge ions were rejected. The parents ions once fragmented were excluded for next 50s with exclusion mass width of ±10 ppm. The AGC target values for MSn were 10000 and 50000 for Ion trap and FTMS respectively. 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 PlasmoDB

28

. Sequences of known

contaminants were added into this database. Carbamidomethyl (C), Deamidation (NQ) and monomethyl and dimethyl (R) were set as variable modifications. Since arginine methylation hinders with trypsin digestion, five missed cleavages were allowed. The resultant identified peptides were validated using Percolator at 5% False Discovery Rate (FDR) (q value

Proteomic Identification and Analysis of Arginine-Methylated Proteins

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