Systematic Identification of the Lysine Succinylation in the Protozoan

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Systematic Identification of the Lysine succinylation in the Protozoan Parasite Toxoplasma gondii Xiaolong Li, Xin Hu, Yujing Wan, Guizhen Xie, Xiangzhi Li, Di Chen, Zhongyi Cheng, Xingling Yi, Shaohui Liang, and Feng Tan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr500992r • Publication Date (Web): 06 Nov 2014 Downloaded from http://pubs.acs.org on November 11, 2014

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Journal of Proteome Research

Systematic Identification of the Lysine succinylation in the Protozoan Parasite Toxoplasma gondii Xiaolong Li1,2‡, Xin Hu2‡, Yujing Wan3, Guizhen Xie3, Xiangzhi Li3, Di Chen4‡, Zhongyi Cheng5, Xingling Yi6, Shaohui Liang3*, Feng Tan3*

1

Department of laboratory medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035, P. R. China

2

School of Medical laboratory science and school of life science, Wenzhou Medical University, Wenzhou, Zhejiang 325035, P. R. China

3

Department of Parasitology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, P.R.China

4

School of the First Clinical Medical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, P.R.China

5

Translational Medcine Advanced Institute, Tongji University, Shanghai 200092, P.R.China

6

Jingjie PTM Biolabs (Hangzhou) Co. Ltd, Hangzhou, Zhejiang 310018, P.R.China

1

ACS Paragon Plus Environment


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KEYWORDS Lysine succinylation, succinylome, lysine succinylation motif, Toxoplasma gondii, tachyzoite

ABSTRACT

Lysine succinylation is a new posttranslational modification (PTM) identified in histone proteins of Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa. However, very little is known about their scope and cellular distribution. Here, using LC-MS/MS to identify parasite peptides enriched by immunopurification with succinyl-lysine antibody, we produced the first lysine succinylome in this parasite. Overall, a total of 425 lysine succinylation sites occurred on 147 succinylated proteins were identified in extracellular Toxoplasma tachyzoites, which is a proliferative stage that results in acute toxoplasmosis. With the bioinformatics analysis, it is shown that these succinylated proteins are evolutionarily conserved and involved in a wide variety of cellular functions such as metabolism, epigenetic gene regulation, and exhibit diverse subcellular localizations. Moreover, we defined five types of definitively conserved succinylation site motifs and the results imply that lysine residue of a polypeptide with lysine on the +3 position and without lysine at the -1 to +2 position is a preferred to be substrate of lysine succinyltransferase. In conclusion, our findings suggest that lysine succinylation in

2


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Toxoplasma involves in a diverse array of cellular functions although the succinylation occurs at a low level.

INTRODUCTION

Protein posttranslational modification (PTM) referred to a dynamic and reversible protein chemical modification after translation plays an important role in protein processing and maturing process, which can change the physicochemical properties of protein, influence the space conformation and stability of protein.1, 2 Among all the amino acids, lysine is a frequent target to be modified because of its role in constructing the spatial structure of proteins and regulating the protein functions. In recent years, mounting evidence indicates that lysine PTM, including methylation,3 ubiquitination,4 acetylation5,

6

and succinylation,7,

8

is an efficient biological

mechanism for broadening the functions of protein and controlling the protein activities.

Compared with both lysine methylation and acetylation, lysine succinylation was determined to promote more substantial transformation to the chemical properties of a protein because succinylation can transfer a bigger structural moiety. Importantly, succinylation occurred on a lysine residue can induces charge status to transform from +1 to −1 under physiological pH condition,9 which in turn facilitate the adjustment of the structure and functions of substrate proteins. As a result, there is possibility that

3



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the more conspicuous structural alteration owing to lysine succinylation could promote more remarkable changes in protein structure and function.

Toxoplasma gondii is one of obligate intracellular protozoan parasites in the phylum Apicomplexa which can parasitize in almost all kinds of nucleated cells of animals and humans. T. gondii infection can cause toxoplasmosis in humans, and it is estimated that nearly 30% of the human population in the world is chronically infected with T. gondii.10 The life cycle of Toxoplasma is complex, with both asexual and sexual stages. Sexual life cycle stage occurs only within feline intestinal epithelium. Asexual life cycle stage, however, can pass through mammalian hosts involving in the developmental switching between two infectious stages: tachyzoites and bradyzoites.11 Chronic infection with latent bradyzoite cysts is asymptomatic in immunocompetent individuals. However, upon host immunosuppression (due to chemotherapy, organ transplantation, or HIV infection) the parasite reconverts into its proliferative tachyzoite form, which causes severe tissue damage ultimately result in organ failure and death.12

In this study, the systematic identification of the lysine succinylome of T. gondii is presented based on an integrated proteome-wide method. Overall, we identified 425 unique lysine succinylation sites identical to 147 succinylated proteins with diverse cellular localizations and biological functions. Moreover, five unique motifs were found through a bioinformatics analysis of the sequences flanking on each side of the

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succinylation sites. To our knowledge, these results provide the first comprehensive view of the succinylome of T. gondii.

MATERIALS AND METHODS

Cell/Parasite Culture and Lysate Preparation

Four 150 cm2 flasks of immortalized human foreskin fibroblast cells (hTERT) monolayers were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) at 37 ℃ with 5% CO2 in a humidified incubator. Confluent hTERT were infected with T. gondii RH strain tachyzoites. After infection, the cells infected with parasites were maintained in DMEM containing 5% heat-inactivated FBS. When approximately 95% of the infected cells had been lysed, the extracellular tachyzoites were harvested and separated from host cells by passage through 23- and 25-gauge syringe needles. Then, the tachyzoites were purified from host cell debris using 3.0-µm Nuclepore filters (Whatman, GE healthcare). Once no intact human cells were visible, the lysate containing intact tachyzoites was washed twice with cold sterile phosphate-buffered saline (PBS) to remove host cell contaminants.

Protein Extraction and Digestion

The parasites pellets were resuspended and lysed in lysis solution composed of 8 M urea, 5 mM dithiothreitol (DTT), 2 mM EDTA, and 1% (v/v) protease inhibitor

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cocktail (Protease Inhibitor Cocktail Set Ⅲ; Callbiochem) and then sonicated with 18 short bursts of 10 s followed by intervals of 10 s on ice. Unbroken debris was removed by centrifugation for 20 min at 4 ℃ and 16,000 × g and protein concentration in the supernatant was confirmed with the 2-D Quant kit (GE Healthcare) in accordance with the manufacturer's protocol. Afterwards, the proteins were precipitated with 20% trichloroacetic acid overnight at 4 ℃. The resulting precipitate was desalted for 3 times with ice-cold acetone by centrifugation for 10 min at 22,000 × g. The air-dried pellet was resuspended in 100 mM NH4HCO3 and digested overnight with trypsin (Promega) at an enzyme : substrate ratio of 1:50 at 37 ℃. The digested peptides were collected and reduced with 5 mM DTT at 56 ℃ for 45 min. Reduced peptides were then alkylated with 15 mM iodoacetamide at room temperature (RT) in the dark for 30 min. The reaction was stopped with 30 mM cysteine for 20 min at RT. To ensure protein digested completely, additional trypsin at an enzyme : substrate ratio of 1:100 was added and incubated for 4 h more. The digested peptides were dried in a SpeedVac (Thermo Scientific) and store at -80℃.

Western Blotting

Western blotting assays were performed using protein lysates from tachyzoites by 12% SDS-PAGE. After transferred to nitrocellulose membrane (Millipore Corp), the membranes were incubated in blocking buffer (0.05% Tween 20 and 5% nonfat milk powder in PBS). Succinylated lysines were detected using rabbit-derived polyclonal

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anti-succinyl lysine antibodies (PTM Biolabs) diluted in blocking buffer at 1:1,000 overnight at 4℃. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Sigma) diluted at 1:2,000 and chemiluminescence substrate for detection (Sigma).

Enrichment of Succinylated Lysine Peptide

Enrichment

of

lysine

succinylated

peptides

was

implemented

by

immunoprecipitation. Briefly, the trypsin digestion was redissolved in NETN buffer (100 mM NaCl, 50 mM Tris, 1 mM EDTA, 0.5% Nonidet P-40, pH 8.0) and incubated with anti-succinyl lysine antibody agarose conjugated beads (PTM Biolabs) at 4℃ overnight with gentle end-to-end rotation. After incubation, the beads were carefully washed 3 times with NETN buffer, twice with ETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 8.0) and 3 times with equivalently purified water. The bound peptides were eluted with 1% trifluoroacetic acid (TFA) and dried under a vacuum. The eluted peptides were cleaned with C18 ZipTips (Millipore) in accordance with the manufacturer's instructions followed by HPLC/MS/MS analysis.

LC-MS/MS Analysis

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was carried out as follows. The immunoprecipitated peptides were resuspended in buffer containing 2% acetonitrile and 0.1% formic acid. After centrifugation, the supernatant was separated onto an Acclaim PepMap 100 C18 trap column (15 cm long with 50 7



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µm i.d., Dionex) by EASY nLC1000 nano-UPLC (Thermo Scientific). Sequently, the peptides were eluted using a 34 min linear gradient at 300 nl / min onto an Acclaim PepMap RSLC C18 analytical column (Dionex). The mobile phase was changed from 5% to 30% elution buffer (EB, 80% acetonitrile, 0.1% formic acid), followed by a 2 min to 40% EB, 2 min to 80% EB, and finally maintained at 80% EB for 4 min.

The peptides were subjected to a NanoSpray Ionization (NSI) source followed by MS/MS in Q Exactive (Thermo Scientific) coupled online to the UPLC. Intact peptides were detected at a resolution of 70,000 in the Orbitrap before peptides were selected using 25% NCE with 4% stepped NCE for MS/MS analysis. Ion fragments were detected at a resolution of 17,500 in the Orbitrap. Alternation between one MS scan followed by 15 MS/MS scans was applied in a data-dependent manner for the top 15 precursor ions above a threshold ion count of 4 × 104 in the MS survey scan with 2.5 s dynamic exclusion. The electrospray voltage applied was set to 1.8 kV. Automatic gain control (AGC) was utilized to prevent overfilling of the ion trap and 2 × 105 ions were accumulated to generate of MS/MS spectra. For MS scans, the m / z scan range was 350 ~ 1800 Da.

Database Search

The protein and succinylation sites were identified using MaxQuant software (v. 1.0.13.13) and Andromeda search 172 engine (v. 1.3.0.5). The MS/MS data were searched sequentially against the T. gondii protein subset of Uniprot database (26518 8


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sequences, http://www.ebi.ac.uk/uniprot/) sequence and ToxoDB 11.0 (8397 sequences, www.toxodb.org), and concatenated with a reverse decoy database and protein sequences of common contaminants. Because trypsin/P belongs to a cleavage enzyme, there was allowed up to three missing cleavages, four modifications per peptide and five charges in the search of the MS/MS data. The mass error was set to ± 4 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on cysteine was specified as a fixed modification and oxidation on methionine, succinylation on both lysine and protein N-terminus were specified as variable modifications. The false discovery rate (FDR) thresholds were specified to be 0.01 for modification sites, peptides and proteins. The minimum length of peptide was set at 7. All the other parameters in MaxQuant analysis were set to default values (“first search” set as none, “main search ppm” set as 4.5, “min. score for modified peptides” set as 40, and “min. delta score for modified peptides” set as 17). Lysine succinylation sites identified with a localization probability of less than 0.75 were removed.

Bioinformatics Analysis

Protein Annotation.

Gene Ontology (GO) annotation proteome was derived from

the ToxoDB 11.0 and UniProt-GOA database (http://www.ebi.ac.uk/GOA/). When a single peptide was found to be matched to two or more different proteins, manual inspection of these data from ToxoDB 11.0 was performed to determine the protein from which the peptide was likely derived. If identified lysine succinylation substrates

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were not annotated by ToxoDB 11.0 and UniProt-GOA database, the InterProScan soft then could be used to annotate protein’s GO function based on protein sequence alignment method. Meanwhile, lysine succinylated proteins were further classified by GO annotation based on three categories: biological process, molecular function and cellular component.

KEGG Pathway Annotation.

Kyoto Encyclopedia of Genes and Genomes

(KEGG) database was used to annotate protein pathway. Firstly, KEGG online service tools KAAS was used to annotate protein’s KEGG database description. Then annotation result was mapped on the KEGG pathway database using KEGG online service tools KEGG mapper. Cello was used for subcellular localization predication.

For enrichment or

GO/KEGG Pathway Functional Enrichment Analysis.

depletion (right-tailed test) of specific annotation terms among members of resulting protein clusters. Fisher’s exact test was used to obtain p values. In any of the clusters, any terms having p values below 0.05 were treated as significant.

Analysis Of Sequence Model Around Succinylation Sites.

The sequences models

constituted with amino acids in specific positions of succinyl-13-mers (6 amino acids upstream and downstream of the succinylation site) in all protein sequences were surveyed using Motif-x. Meanwhile, all T. gondii database protein sequences were used as background database parameter, other parameters were set as default.

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Motif-Based Clustering Analysis.

All the lysine succinylation substrates

categories obtained after enrichment were collated along with their p values, and then filtered for those categories which were at least enriched in one of the clusters with p value < 0.05. This filtered p value matrix was transformed by the function x = −log10 (p value). Finally these x values were z-transformed for each category. These z scores were then clustered by one-way hierarchical clustering (Euclidean distance, average linkage clustering) in Genesis. Cluster membership was visualized by a heat map using the “heatmap.2” function from the “ggplots” R-package.

RESULTS AND DISCUSSIONS

Proteome-Wide Analysis of Lysine Succinylation Sites and Proteins in Toxoplasma

Lysine succinylation, a new acylation type among various PTM, has been thought to play roles in the regulation of protein function in diverse ways in both prokaryotic and eukaryotic cells.13 In contrast, the succinylome in Toxoplasma gondii has not yet been reported. The genome database of T. gondii was collated and collected about a decade ago,14 which promote the systematic analysis of the lysine succinylated sites and proteins in this species. In this study, we combined lysine succinylated peptides enrichment with highly sensitive mass spectrometry and bioinformatics tools together. In order to obtain a detailed view of lysine succinylation sites in T. gondii, the proteins were isolated from extracellular tachyzoites that have freshly egressed from 11



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host cells. After digestion with trypsin, the succinylated peptides were enriched by affinity purification using succinyl-lysine antibodies. Then, the enriched succinylated peptides were surveyed by LC-MS/MS analysis and the obtained MS/MS data were matched to both ToxoDB and Uniprot database. The mass error was set to ±4 ppm for precursor ions.

Using the above-described approach, a total of 425 lysine succinylation peptides with a peptide score greater than 40 (Table S1 in the Supporting Information) were identified in Toxoplasma tachyzoites. The 425 peptides, which showed different abundances depending on length, occurred on 147 succinylated proteins possessing different succinylated sites from 1 to 17. Of the total of 147 succinylated proteins identified, approximately 41% (60/147) proteins possessed a single succinylated site, 20% (29/147) possessed two succinylated sites, 10% (16/147) carried three identified sites, and the average degree of succinylation was 2.9 (426/147). Notably, there are 29 proteins that possessed five or more succinylated sites. The most extensively succinylated protein is the heat shock protein (HSP), which succinylated at up to 17 independent lysine residues.

Characterization of Lysine Succinylome of Toxoplasma

To identify the presence of succinylated protein in Toxoplasma, tachyzoite lysate was prepared for Western blot using an antibody recongnizing succinyl-lysine. As expected, the data determined that a number of proteins with a wide range of 12


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molecular masses are succinylated (Figure 1A). To better understand the characterization of succinylated proteins in Toxoplasma, we annotated all of the identified succinylated proteins with the GO functional classification. The classification results in terms of GO revealed that succinylation occurred on diverse proteins involved in biological process and molecular function, indicating that succinylation is an important PTM in T. gondii. On the basis of results for both biological process and molecular function, the largest class of succinylated proteins was catalytic enzymes found in association with molecular function, accounting for 27% of the all annotated succinylated proteins. The second largest class consisted of proteins involved in metabolic process, accounting for 19% of all of the identified proteins. Another large succinyl protein class included binding proteins, accounting for 16% of all identified proteins (Figure 1B). These findings are consistent with previous results in bacteria and eukaryotic cells,13 suggesting the essential regulatory roles of succinylated proteins in cells.

In addition, to confirm which functional categories are preferred targeted for lysine succinylation, enrichment analysis of the succinylation was evaluated (Figure 1C and Table 1). A wide range of metabolic processes related to glucose, hexose, monosaccharide and single-organism carbohydrate were observed to be significant enrichment of succinylated proteins. Meanwhile, enrichment of succinylated proteins involved in oxidoreductase activity was also observed. To obtain a more comprehensive understanding of the metabolic processes involved, KEGG enrichment 13



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analysis was further carried out (Figure 1D and Table 2). Our results showed that multiple metabolic pathways were highly rich in succinylome, associating with the citrate cycle, carbon metabolism, metabolic pathways, biosynthesis of secondary metabolites. Overall, these finding indicate a vital role of lysine succinylation in most fundamental cellular processes of T. gondii.

Lysine Succinylation on Mitochondrial and Apicoplast Proteins in Toxoplasma

To date, there were no previous reports on subcellular distribution and compartmentalization of succinyl-CoA, although acetyl-CoA was found to spread in separate mitochondria and nonmitochondrial subcellular organelles.15, 16 Therefore, the subcellular location of the succinylated proteins was also analyzed, and the classification results showed that 26% (38/147) of the succinylated proteins were located in mitochondria, 24% (35/147) distributed in the chloroplast, 23% (33/147) located in the nuclear, and 19% (28/147) proteins are cytoplasmic proteins, as shown in Figure 2A and Supporting Information Table S2.

It is known that succinyl CoA and succinate are mainly derived from the mitochondria in the process of the tricarboxylic acid cycle or by odd-numbered fatty acid oxidation, the succinylation protein is thus biased to occur on more mitochondrial proteins in some eukaryotic organisms. Recently, it is reported that the proportion of succinylation occurring on mitochondrial proteins in yeast, HeLa cells and mouse liver is approximately 8%, 45% and 70%, respectively.13 Similarly, in 14


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present study, we demonstrated that the succinylation protein is biased to occur on mitochondrial proteins in T. gondii. It is noteworthy that the proportion of succinylation occurring on mitochondrial proteins is discrepant depended on various eukaryotic organisms. There is a reasonable explanation that MS analysis is more readily to detect more abundant proteins, such as mitochondrial proteins, which account for a larger proportion in HeLa cells and mouse liver.

Although the proportion of succinylated mitochondrial proteins in Toxoplasma was significantly smaller than in HeLa cells and mouse liver, we found a significant number of succinylation proteins were located in chloroplast, a subcellular organelle termed apicoplast in apicomplexans. For instance, after performance of KEGG pathway enrichment analysis, we found that nearly every enzyme involved in the tricarboxylic acid cycle metabolic pathway was distributed in both mitochondria and apicoplast (Figure 2B and Supporting Information Figure S1 and S2). Because most proteins targeted to the apicoplast lumen are modified by PTM subsequent to nuclear-encoded,17-20 it is suggested that succinylation occurring on apicoplast proteins may regulate a wide range of functional cell processes. Moreover, apicoplast is essential to parasite survival and involved in the synthesis of fatty acids, isoprenoids, iron-sulfur clusters and heme.21 Importantly, these proteins and pathways have no homologues in its host cells. As a result, the lysine succinylation sites in apicoplast protein may hold great promise in the development of new therapeutic drugs with minimum side effects to the infected host.22, 23 15



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Lysine Succinylation on Cytoplasmic Proteins in Toxoplasma

As described in previous studies, succinate can span the mitochondrial membrane and can be produced in the cytoplasm as a by-product of α-ketoglutarate-dependent enzymes.24 In this research, we found many succinylation proteins were also distributed in cytoplasm.

Glyceraldehyde-3-phosphate dehydrogenase I (GAPDH I), found in the parasite cytoplasm, is succinylated on two lysines (202SAGVNIIPASTGAAKsuAVGK and su 47YDSVHGHYPGEVSHK DGK,

Ksu indicates succinylated lysine, Supporting

Information Figure S3), and K216su is conserved with a succinylation marker on the E. coli, mouse and human GAPDH.13 Toxoplasma phosphoglycerate kinase (PGK1) is succinylated on K130 (123FHIEEEGKsuGVDEQGNK), which is analogous to a succinylated lysine in human and mouse PGK1.13 In addition, a lot of other cytoplasmic proteins, including actin depolymerizing factor (ADF) and actin itself, were found to be succinylated in T. gondii. A lysine succinylation site, K90su (84MTYASSKsuDALLK), on ADF matches a succinylation site on the human and K61su (51DCYVGDEAQSKsuR) on actin is conserved in the mouse.13

The abundance of lysine succinylation on nonmitochondrial proteins indicated that succinyl CoA, succinate, or another succinyl-metabolite promotes succinylation in the cytoplasm and other subcellular organelles, which is associated with more biological

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processes, including gene transcription, protein translation, and various metabolic pathways.

Lysine Succinylation on Histone Proteins in Toxoplasma

With a complex life cycle, it is necessary for Toxoplasma to regulate gene expression in response to a variety of stimuli with a precise and fast regulatory system, such as epigenetic gene regulation. Mounting evidence suggests that PTM of histones plays a key role in epigenetic gene regulation.25 Obviously, understanding this epigenetic process and its roles in cellular physiology may increase the potential of the development of new antiparasitic drugs. Based on several mutagenesis strategies, it has been indicated that succinylation can function in chromatin structure, nucleosome interaction with DNA, transcriptional regulation and rDNA silencing,7 though the regulatory potential of histone marks is not completely elucidated in histone lysine succinylation.

Core histones in T. gondii include a single copy of H2A, H3 and H4, as well as 2 isoforms of H2B (H2Ba and H2Bb).25 Both H3 and H4 are highly conserved, but H2A and H2B are more divergent. The succinylation on histone H3 and H4 in eukaryotic species has previously been documented.7 Both H3K122su (116RVTIMPKsuDIQLAR) and H4K31su (24DNIQGITKsuPAIR) were confirmed in our investigation (Figure 3 and Supporting Information Figure S4), which is consistent with recently published results in T. gondii histone succinylation,25 indicating the two succinylation sites are 17



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highly conserved, while the failure to detect H4K16su may be owing to the transient features of succinylation modification or a limitation connected with the affinity of anti-succinyl antibody used for peptide enrichment in this study. Intriguingly, H3K56su (54YQKsuSTDLLIR) found in this study is a novel histone succinylation mark that has not yet to be described in apicomplexans, although this has been reported in T. gondii as a formylated peptide,25 an additional PTM emerged from DNA oxidative damage.26 Besides, both H3K122 and H4K31 were also previously identified to be formylated.25

In previous research, the histone code of T. gondii tachyzoites was enriched with standard acid extraction procedure followed by 2D LC-MS/MS analysis.25 However, neither H2A or H2B was found with succinylation modification. There are several possible reasons as follows: (i) the peptides corresponding to H2A and H2B was less abundance; (ii) these proteins were wasted artificially during sample preparation; (iii) acid extraction changes the accessibility of proteins. In this study, while no succinylation was identified on histone H2A, we identified five sites of Ksu in Toxoplasma

histone

H2B,

(100HAVSEGTKsuAVTK, su 35VLK QVHPETGISK)

of

which

four

su 64IASEAGK LCK,

sites

are

on

histone

H2Ba

su 91LVLPGELAK HAVSEGTK

and

and one site on H2Bb variant (105HAVSEGTKsuAVTK)

possessing the same amino acid sequences with H2BaK107su (Figure 3). Either H2BaK70su or H2BaK99su is the unique lysine succinylation site in Toxoplasma that have not been reported. In contrast, both H2BaK107su and H2BaK37su have been 18


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found in a variety of eukaryotic species.7 It is known that the H2Ba is upregulated in tachyzoites stage and the H2Bb is expressed in whole sexual stages but neither in tachyzoites or bradyzoites.27 Therefore, we argue that the lysine succinylation on histone core could be a critical event for the growth and differentiation of the parasite, as well as stage-specific adaptation to different environments during transition between hosts, although the mechanism of succinylation on histone core remains unknown.

Motifs Analysis for Identified Lysine Succinylated Peptides

To evaluate the nature of the succinylated lysine in Toxoplasma, we used Motif-X program, a software developed to extract overrepresented patterns from any sequence data set, to search the sequence motifs in all the identified succinylated lysines. As shown in Figure 4A and Table 3, a total of 5 definitively conserved succinylation site motifs were defined on 146 unique sites accounting for 34% of sites identified according to the criteria of specific amino acid sequence from 6 amino acids upstream and downstream of the succinylated lysine. These are XXXXXXKsuXXXKXX, XXIXXXKsuXXXXXX,

XXXXXLKsuXXXXXX,

XXXXXXKsuGXXXXX,

XXXXXQKsuXXXXXX (X indicates a random amino acid residue). The sequence logos do not show a strong bias for a particular amino acid. However, isoleucine (I), leucine (L) and glutamine (Q) occurred most frequently at the upstream of the Ksu (I was observed on the third position toward the N terminus, L and Q was observed on

19



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Page 20 of 40

the sixth position toward the N terminus), while lysine (K) and glycine (G) are found frequently downstream of the Ksu (K was observed on the third position toward the C terminus, G was observed on the sixth position toward the C terminus). These biases for these amino acids may represent a genuine preference or may be attributed to the sequence bias of anti-succinyl lysine antibodies used for selectively enrichment of succinylated peptides, as were previously described for acetyl-lysine enrichment in T. gondii.28

In order to determine whether there is significant frequency of specific amino acids flanking succinylated lysine site, these results were further demonstrated by a logo reflecting relative frequency of amino acids in specific positions of succinyl-13-mers (6 amino acids upstream and downstream of the modification site) compared to that of nonsuccinyl-13-mers

(6

amino

acids

upstream

and

downstream

of

the

non-modification site). Indeed, these results demonstrated by intensity map is in accordance with these results above. Furthermore, as shown in Figure 4C, because of the lowest frequency of K in positions -1 to +2 in the motifs, It is reasoned that lysine residue of a polypeptide with K on the +3 position and without K at the -1 to +2 position is a preferred to be substrate of lysine succinyltransferase.

Overlap Between Lysine Succinylation and Acetylation

Lysine acetylation has been recently identified in T. gondii.28, 29 Consequently, we compared the succinylation sites identified in our study to previously determined 20


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acetylation sites in Toxoplasma. We found that 22 succinylation sites on 17 proteins were acetylated at the same position (Figure 5 and Supporting Information Table S3). Of which K164 and K534 on FAD Malate-dehydrogenase (MDH-FAD) identified in this study as lysine succinylated are also acetylated. Other overlap between succinylation and acetylation found in two lysine residues of one protein includes ADF, 14-3-3 protein and eukaryotic porin protein. Besides, succinylation of K112 on histone H2B variant and K31 on histone H4 are acetylated at the same site. These complicated overlap between the two important PTMs indicated that cellular metabolism in Toxoplasma is a highly dynamic process.

CONCLUSION

Combining high affinity enrichment of lysine succinylated peptides with high sensitive mass spectrometry and bioinformatics tools, we provided the first in-depth insight of the lysine succinylome for a single-celled eukaryote and parasite. We identified 425 lysine succinylation sites occurring on 147 succinylated proteins in Toxoplasma tachyzoites. Moreover, with the extensive characterization of the succinylome, we observed that succinylation occurred on a large scale of Toxoplasma proteins targeting a broad range of functions ranging from control of metabolic process to biological regulation, and the succinylated proteins are distributed in different cellular compartments, suggesting that protein succinylation is likely to be vital in regulating T. gondii physiology. Particularly, the lysine succinylation of

21



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proteins related to catalytic activity and metabolic process may play more important physiological roles. Indeed, our data provide a significant resource to facilitate the illumination of the role of succinylation and the abundance of succinylation in T. gondii, which will increase our understanding of lysine succinylation in the cellular physiology and metabolites biosynthesis of T. gondii and other members of the Apicomplexa phylum.

22


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Tables Table 1 Enrichment analysis of the succinylated protein classified by GO annotation in T. gondii

GO Terms

Mapping

Background

All

All

Fold

mapping

background

enrichment

Fisher' exact Test P value

Enrichment of biological process glucose metabolic

3

38

10

1312

10.35789

0.02587

3

40

10

1312

9.84

0.0285

3

41

10

1312

9.6

0.02986

3

48

10

1312

8.2

0.04005

197

13

1999

4.683327

0.00393

process hexose metabolic process monosaccharide metabolic process single-organism carbohydrate metabolic process Enrichment of molecular function oxidoreductase

6

activity

23



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Table 2. KEGG pathway enrichment analysis of the identified succinylated proteins in T. gondii

KEGG Pathway

Mapping

Background

All

All

Fold

Mapping

Background

enrichment

Fisher' exact Test P value

Citrate cycle

12

73

49

3115

10.4501

6.14E-09

Carbon metabolism

17

198

49

3115

5.458153

1.44E-08

Metabolic pathways

34

1040

49

3115

2.078297

4.85E-07

Biosynthesis of

21

411

49

3115

3.248175

7.87E-07

9

122

49

3115

4.689696

4.45E-04

5

50

49

3115

6.357143

0.0068

Pyruvate metabolism

5

53

49

3115

5.997305

0.008359

Glyoxylate and

4

28

49

3115

9.081633

0.008586

4

33

49

3115

7.705628

0.01355

3

13

49

3115

14.67033

0.01628

Lysine degradation

3

15

49

3115

12.71429

0.02149

Biosynthesis of amino

7

159

49

3115

2.798742

0.03339

secondary metabolites Oxidative phosphorylation Valine, leucine and isoleucine degradation

dicarboxylate metabolism 2-Oxocarboxylic acid metabolism Phenylalanine metabolism

acids

24


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Table 3. Motif Analysis for Identified the Succinylated Lysine Peptides in T. gondii

Motif

Foreground

Background

Fold

Motif

*

score

Matches

Size

Matches

Size

increase

XXXXXXKsuXXXKXX*

2.31

38

420

244

4211

1.56

XXIXXXKsuXXXXXX

2.1

25

382

153

3967

1.7

XXXXXLKsuXXXXXX

2.09

43

357

314

3814

1.46

XXXXXXKsuGXXXXX

2.25

34

314

239

3500

1.59

XXXXXQKsuXXXXXX

2.46

26

280

170

3261

1.78

The X denotes an any amino acid

25



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Figure captions

Figure 1. Characterization of lysine succinylome of T. gondii. (A) Western blot analysis of tachyzoite lysate using succinyl-lysine antibody demonstrate the presence of succinylated proteins. (B) Gene Ontology functional classification of the identified succinylated proteins. (C) Enrichment analysis of the succinylated proteins based on the classification of GO annotation in terms of molecular function (red bars) and biological process (green bars) (p < 0.05). (D) KEGG pathway enrichment analysis (p < 0.05).

Figure 2. Subcellular location of lysine succinylome of T. gondii. (A) Gene Ontology functional classification of the identified succinylated proteins based on the subcellular location. (B) The subcellular location of succinylation of enzyme involved in the Tricarboxylic Acide Cycle. The enzymes shown are pyruvate dehydrogenase E1 component subunit beta (PDH-E1II), pyruvate dehydrogenase complex subunit (PDH-E3II), citrate synthase I (CS1), aconitate hydratase (ACN), isocitrate dehydrogenase (IDH), dihydrolipoamide S-succinyltransferase (DLST), succinyl-CoA ligases subunit beta (SUCL2), succinate dehydrogenase (SDHA), fumarate hydratase (FH), malate dehydrogenase (MDH).

Figure 3. Comparison of succinylated residues in histone proteins of T. gondii (Tg) with that of S. cerevisiae (Sc), D. melanogaster (Dm), M. musculus (Mm), and H.

26


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sapiens (Hs). Red sequences indicate the succinylation sites. Numbers below the sequences represent the amino acide position.

Figure 4. Characterization of succinylated peptides. (A) Probability sequence motifs of Toxoplasma succinylation sites consisting of 12 residues surrounding the targeted lysine residue using Motif-X. Five significantly enriched succinylation site motifs were identified. (B) Number of identified peptides possessing succinylated lysine in each motif. (C) Heat map showing enrichment (red) or depletion (green) of amino acids in specific positions flanking the succinylated lysine in Toxoplasma.

Figure 5. Venn diagram outlined the overlap between succinylation and acetylation in T. gondii. (A) The overlapped sites between succinylation and acetylation. 22 succinylation sites identified were also acetylated at the same position. (B) The overlapped proteins between succinylation and acetylation. 17 succinylation proteins identified were also acetylated.

ASSOCIATED CONTENT

Supporting Information

Figure S1, The spectra of citrate synthase K133 (A) and K550 (B); Figure S2, The spectra of isocitrate dehydrogenase K226 (A), K386 (B) and K429 (C); Figure S3, The spectra of GAPDH K61 (A) and K216 (B); Figure S4, The spectra of histone H3K56 (A), H3K122 (B) and H4K31 (C); Table S1, all T. gondii lysine succinylated

27



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peptides and proteins identified by LC-MS/MS, PEP indicates the maximal posterior error probability; Table S2, Subcellular localization classification of identified acetylated proteins; Table S3, Identified T. gondii proteins overlapped between succinylation and acetylation. This material is available free of charge via the Internet at http://pubs.acs.org. Abbreviations list PTM, posttranslational modification; hTERT, immortalized human foreskin fibroblast cells; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes

AUTHOR INFORMATION Corresponding Author * Shaohui Liang. E-mail: [email protected]; Tel: +086 577 86689860; Fax: +086 577 86699561

*Feng Tan. E-mail: [email protected]; Tel: +086 577 86689860; Fax: +086 577 86699561

Notes The authors declare no competing financial interest. Author Contributions

28


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‡

These authors contributed equally. All authors have given approval to the final

version of the manuscript.

ACKNOWLEDGMENT

This work was supported by grants from the Natural Science Foundation of Zhejiang Province (LY12H19004) and the Commonweal Technology Application Project Program of Zhejiang Province (2014C33161) to F.T. and the Natural Science Foundation of Zhejiang Province (LQ14H190003) to X.H, as well as National Natural Sciences Foundation to CZ (31271362). We are grateful to Dr. William J. Sullivan, Jr. (Indiana University School of Medicine, IN) for providing the hTERT cell strain.

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Figure 1 Characterization of lysine succinylome of T. gondii. (A) Western blot analysis of tachyzoite lysate using succinyl-lysine antibody demonstrate the presence of succinylated proteins. (B) Gene Ontology functional classification of the identified succinylated proteins. (C) Enrichment analysis of the succinylated proteins based on the classification of GO annotation in terms of molecular function (red bars) and biological process (green bars) (p < 0.05). (D) KEGG pathway enrichment analysis (p < 0.05). 101x70mm (300 x 300 DPI)



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Figure 2 Subcellular location of lysine succinylome of T. gondii. (A) Gene Ontology functional classification of the identified succinylated proteins based on the subcellular location. (B) The subcellular location of succinylation of enzyme involved in the Tricarboxylic Acide Cycle. The enzymes shown are pyruvate dehydrogenase E1 component subunit beta (PDH-E1II), pyruvate dehydrogenase complex subunit (PDHE3II), citrate synthase I (CS1), aconitate hydratase (ACN), isocitrate dehydrogenase (IDH), dihydrolipoamide S-succinyltransferase (DLST), succinyl-CoA ligases subunit beta (SUCL2), succinate dehydrogenase (SDHA), fumarate hydratase (FH), malate dehydrogenase (MDH). 62x25mm (300 x 300 DPI)


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Figure 3 Comparison of succinylated residues in histone proteins of T. gondii (Tg) with that of S. cerevisiae (Sc), D. melanogaster (Dm), M. musculus (Mm), and H. sapiens (Hs). Red sequences indicate the succinylation sites. Numbers below the sequences represent the amino acide position. 115x78mm (300 x 300 DPI)



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Figure 4 Characterization of succinylated peptides. (A) Probability sequence motifs of Toxoplasma succinylation sites consisting of 12 residues surrounding the targeted lysine residue using Motif-X. Five significantly enriched succinylation site motifs were identified. (B) Number of identified peptides possessing succinylated lysine in each motif. (C) Heat map showing enrichment (red) or depletion (green) of amino acids in specific positions flanking the succinylated lysine in Toxoplasma. 130x112mm (300 x 300 DPI)


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Figure 5 Venn diagram outlined the overlap between succinylation and acetylation in T. gondii. (A) The overlapped sites between succinylation and acetylation. 22 succinylation sites identified were also acetylated at the same position. (B) The overlapped proteins between succinylation and acetylation. 17 succinylation proteins identified were also acetylated. 24x7mm (300 x 300 DPI)



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Abstract Graphic 46x25mm (300 x 300 DPI)


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Systematic Identification of the Lysine Succinylation in the Protozoan

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