Proteomic Analysis of Plasmodium falciparum Schizonts Reveals

Article pubs.acs.org/jpr

Proteomic Analysis of Plasmodium falciparum Schizonts Reveals Heparin-Binding Merozoite Proteins Yan Zhang,†,# Ning Jiang,†,# Huijun Lu,† Nan Hou,‡ Xianyu Piao,‡ Pengfei Cai,‡ Jigang Yin,† Mats Wahlgren,*,§ and Qijun Chen*,†,‡,§ †

Key Laboratory of Zoonosis, Ministry of Education, Jilin University, Xian Da Lu 5333, Changchun 130062, China Institute of Pathogen Biology, Chinese Academy of Medical Sciences, Dongdan San Tiao 9, Beijing 100730, China § Department of Microbiology, Tumour- and Cell Biology, Karolinska Institutet, Nobels väg 16, SE171 71 Stockholm, Sweden ‡

S Supporting Information *

ABSTRACT: The malaria parasite Plasmodium falciparum utilizes host glycosaminoglycans (GAGs) as receptors for erythrocyte invasion and intravascular sequestration. Heparin and heparan sulfate (HS) are GAGs which can block erythrocyte invasion of the P. falciparm merozoite, albeit the molecular mechanisms remain poorly understood. Characterization of these heparin-binding merozoite proteins and key ligands in the host−parasite interplay will lead to a better understanding of the mechanism of erythrocyte invasion by malaria parasites. Here, schizont-derived proteins that bind heparin were enriched by affinity chromatography, and 6062 peptides from 811 P. falciaprum-derived proteins were identified by two-dimensional liquid chromatography−mass spectrometry (LC/LC−MS/MS). The proteins were categorized into 14 functional groups ranging from pathogenesis, protein catabolic process to signal transduction. Proteins with predominant peptide counts were found to mainly originate from the rhoptry organelle of merozoites and the parasitized erythrocyte membrane. The profile of the heparin/HS-binding proteome of P. falciparum suggests they have important functions in the biology of the parasite. KEYWORDS: Plasmodium falciparum, LC/LC−MS/MS, proteome, heparin-binding proteins, function

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INTRODUCTION Plasmodium falciparum is a major cause of high morbidity and mortality in malaria endemic areas, especially in sub-Saharan Africa.1 The pathogenesis of malaria occurs after merozoites invade and multiply within host erythrocytes. The invasion of a red blood cell (RBC) by P. falciparum is a cascade process with the initial attachment of a merozoite to the RBC surface, followed by reorientation and eventual penetration of the erythrocyte membrane, which involves both parasite ligands and RBC receptors.2−4 Merozoite surface associated ligands such as the apical membrane protein-1 (AMA1), the merozoite surface proteins (MSPs) family members, mediate the initial attachment of the parasite to the RBC receptors such as sialic acid, complement receptor 1 (CR1),5,6 and a recently identified receptor basigin.7 The timely release of microneme- and rhoptry-derived ligands such as EBA-175 and the rhoptry proteins such as CLAG3.1 to the erythrocyte surface is also critical for receptor-engagement and successful invasion.8,9 With the development of the parasite inside the RBC (pRBC), P. falciparum expresses parasite-derived surface adhesins and sequesters in the postcapillary venules in order to avoid splenic clearance. Sequestration of pRBCs is to a large extent mediated by the interaction of the parasite-derived adhesin, P. falciparum erythrocyte membrane protein 1 (PfEMP1), with host © 2013 American Chemical Society

vasculatural receptors. PfEMP1 interacts with a variety of host endothelial receptors including CD31, CD36, ICAM-1, and a member of glycosaminoglycan (GAG) family such as heparin sulfate (HS) and chondroitin sulfate A (CSA).10 Heparin and HS are GAGs consisting of repeated disaccharide units comprising a hexuronic acid (HexA) and a D -glucosamine (GlcN), which display an extraordinary structural diversity and different patterns of sulfation. During the biosynthesis of HS, the uronic group may be either a β-Dglucuronic acid (GlcA), its epimer or α-L-iduronic acid (IdoA) which is modified with O-sulfate. N-Sulfated glucosamines (GlcNSO3) may occur either at C3 (GlcNSO33S), C6 (GlcNSO36S), or at both places (GlcNSO33S6S). Similarly, the N-acetylated glucosamines (GlcNAc) may be O-sulfated at C6 (GlcNAc6S).11 In contrast, heparin has a high ratio of IdoA to GlcA with more GlcNSO3 than GlcNAc in molecular constitution. Thus, heparin has a higher degree of sulfation than HS. Heparin is only present in mast cells, whereas HS is present on all human cell surfaces including the RBC.12,13 Heparin/HS are viewed as potential therapeutics for a variety of diseases, and previous work has shown that both heparin and HS can inhibit Received: January 14, 2013 Published: April 8, 2013 2185

dx.doi.org/10.1021/pr400038j | J. Proteome Res. 2013, 12, 2185−2193

Journal of Proteome Research

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merozoite invasion, disrupt P. falciparum rosettes, inhibit endothelial-binding, and release sequestered pRBC into circulation in rat and macaque animal models.14−17 Heparin has previously been used as an adjunct drug in patients with severe malaria, but its application was discontinued due to the occurrence of intracranial bleedings.18−20 On the other hand, a chemically modified heparin with a low anticoagulant activity has been found to inhibit merozoite invasion, disrupt rosette, and desequester pRBCs suggesting that the antiparasitic activities do not depend on the anticoagulant property.16,21,22 Though PfEMP1 and merozoite surface protein 1 (MSP1) have been reported as the targets for heparin/HS in both rosette disruption, desequestration, and invasion inhibition,16,17 the molecular mechanisms of heparin and HS on parasite invasion have been largely unknown. Here, we report the identification and characterization of the heparinbinding proteome presented in schizont-infected P. falciparum erythrocytes. The data to a large extent explain the mechanism of the antimalaria effect of heparin.

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iodoacetamide (IAM) in the dark for 45 min, followed by trypsinization at 37 °C for 16 h. Two batches of tryptic peptides were reconstituted separately with 4 mL of buffer (25 mM NaH2PO4, 25% acetonitrile, pH 2.7) and subjected to LC/ LC−MS/MS analysis. One batch was used in the preliminary run with default parameters to test the system and the efficiency of protein identification. The other batch was run as a formal test. The LC/LC−MS/MS analysis was carried out in combination of strong cation exchange (SCX) and reversed-phase chromatography to achieve two-dimensional separation prior to MS/MS. Briefly, the complex peptide mixture was loaded onto a 4.6 × 250 mm Ultremex SCX column containing 5 μm particles (Phenomenex, CA, USA) for fractionation. The eluted peptides were collected as 10 fractions, which were desalted using Strata X C18 column (Phenomenex) and further vacuumdried, respectively. Each fraction was resuspended in buffer A (2% acetonitrile, 0.1% formic acid) and then transferred to C18 reverse phase columns of Shimadzu LC-20AD nanoHPLC system at a flow rate of 15 μL/min for 4 min for a final separation. The samples were eluted with a linear gradient from 2 to 80% buffer B (98% acetonitrile, 0.1% formic acid) at a flow rate of 0.4 μL/min for 60 min. The peptides were finally electrosprayed into an LTQ Orbitrap Velos (Thermo Fisher, MA, USA) coupled online to the nanoHPLC for an automated MS/MS analysis.

MATERIALS AND METHODS

Collection of P. falciparum Schizonts

The highly synchronous P. falciparum (3D7 line) parasites were cultured in human O+ erythrocytes with 5% AB+Rh+ serum and 0.25% AlbuMAXII according to standard methods.23 The schizonts were purified by gradient Percoll centrifugation (from top to bottom: 40%, 60%, 70%, and 80% Percoll) as described (http://www.mr4.org). The cells at the 40−60% interface, containing mostly schizonts, were collected.

Mass Spectrometry Data Analysis

The obtained mass spectrum data files from the two runs were compared and the peptide data were searched against the P. falciparum 3D7 annotated protein sequence database (PlasmoDB 7.1 released, 5492 sequences, November 18, 2010) using the in-house developed software MaxQuant (version 1.1.1.36).25 The mass tolerance for precursor ions and fragment ions was 20 ppm. Fixed modifications such as cysteine carbamidomethylation and variable modifications such as methioninee oxidation and N-terminal acetylation were searched, and the monoisotopic mass values of the peptides were determined. The maximum missed cleavage was 1, and the false positive rate (FDR) for peptide and protein identification was set as 1%. Peptide identities were chosen to be correct with MaxQuant scores more than 45, and all peptides with scores below this were discarded. At least two unique peptides were required to identify a protein.26 Proteins identified by more than 30 peptides were selected for further characterization. Protein function was assigned based on the P. falciparum database annotation and literature references.

Affinity Purification of Native P. falciparum Proteins with Heparin-Sepharose

The purified schizonts suspended in phosphate buffered saline (PBS) containing a complete cocktail of protease inhibitors were freeze−thawed several times and centrifuged at high speed for 15 min. To test the specificity of heparin-binding of the parasite proteins, the supernatant was divided into 10 fractions and mixed separately with either heparin 5000 (Sigma-Aldrich, CA, USA) or CSA (Sigma-Aldrich) at a final concentration ranging from 2 to 0.1 mg/mL for 30 min at 4 °C. The mixture was then incubated with 20 μL of heparin-sepharose at 4 °C for 2 h, and the beads were washed three times in large volumes of PBST buffer (PBS plus 0.05% Tween-20). Bound proteins were dissolved on a 10% SDS-PAGE and detected by Western-blot using a pool of human anti-P. falciparum sera (diluted 1:500) which has been used to characterize P. falciparum-derived proteins.24 To further fingerprint the polypeptides bound to heparin, the supernatant of parasite lysate was mixed with aliquots of 300 μL of heparin-sepharose and incubated at 4 °C for 2 h. The mixture was washed three times in large volumes of PBST buffer. The bound proteins were eluted by stepwise increased concentrations of 1.0, 1.5, and 2.0 M NaCl.

Expression of Recombinant Proteins for Confirmation of Specific Heparin Binding

For those proteins with a molecular weight of more than 80 kDa, the sequences were divided into 2−5 fragments for generation of recombinant proteins based on two criteria, one was based on the functional domains previously characterized; the other was based on the regions with most peptides located and the protein cleavage characteristics. Different fragments of the selected proteins were amplified from the complementary DNA (cDNA) using specific primers (Supporting Information 1). The amplified fragments were cloned into pGEX-4T-1 or pET32a vectors and expressed in Escherichia coli BL21 (DE3), and the fusion proteins were purified on glutathione−sepharose or His GraviTrap columns (GE Healthcare) respectively according to the protocols provided by the manufacturers.

Sample Preparation and Two-Dimensional Liquid Chromatography with Tandem Mass Spectrometry

Proteins eluted from heparin beads were precipitated by acetone and redissolved in 500 μL of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-HCl, pH8.5), sonicated at 200 W for 15 min. The supernatant containing 100 μg of proteins quantified using a 2-D Quant kit (GE Healthcare, Uppsala, Sweden) was treated with 10 mM dithiothreitol (DTT) at 56 °C for 1 h and alkylated with 55 mM 2186



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Figure 1. Affinity chromatography of heparin-binding proteins. Soluble proteins of P. falciparum in PBS buffer were incubated with heparinconjugated sepharose or sepharose alone (A), and proteins of multiple molecular sizes were pulled down by heparin-sepharose (A, lane 1) but not by sepharose alone (A, lane 2). To further test the specificity, the proteins were incubated with various concentrations of heparin and CSA before incubation with heparin-sepharose. Proteins bound to heparin were resolved in SDS-PAGE and detected with a malaria hyperimmune serum. Results indicate the binding of P. falciparum proteins to heparin-sepharose can be specifically inhibited by heparin (B) in a concentration-dependent manner; CSA does not show any inhibition effect (C).

Figure 2. Identification of proteins by MS/MS. Representative MS/MS spectra of peptides from four selected proteins were identified based on peptide hits. MS/MS spectra show the annotation of fragment ions (b and y ions) of the peptide peak. Panel A shows the MS/MS spectrum of the peptide DFTQTNALTNLPNLDNK of PfRh2. Panel B shows the MS/MS spectrum of the peptide KLSFLS SGLHHLITELK of MSP1. Panel C shows the MS/MS spectrum of the peptide INFNDIQLTMEDEVVR of RON3. Panel D shows the MS/MS spectrum of the peptide ESNTALESAGTSNEVSER of SERA5.

Heparin-Binding Assay with Recombinant Proteins

incubation with heparin-sepharose. The inhibitory effect was detected by Western-blot as described above.

The potential binding of the recombinant proteins to heparin was studied as previously described.27 Soluble recombinant proteins and GST alone (1 μM in PBS buffer) were respectively mixed with hepain-sepharose and uncoupled sepharose 4B (GE Healthcare) at 4 °C for 2 h. The bound proteins were detected by Western-blot using a pool of human anti-P. falciparum sera (diluted 1:500) which has been used to characterize P. falciparum-derived proteins.24 Binding inhibition of recombinant protein to heparin-sepharose by heparin and CSA was also tested. The soluble recombinant proteins (1 μM in PBS buffer) were mixed separately with either heparin or CSA at a final concentration ranging from 0.001 to 100 mg/mL before

Merozoite Invasion Inhibition with GAGs and Merozoite-Derived Heparin-Binding Proteins

Glycosaminoglycans and recombinant proteins were tested for invasion inhibition against P. falciparum parasites according to the reports earlier.16,17 Briefly, synchronized P. falciparum cultures (ring stage) with a parasitemia of 0.5% and a hematocrit of 5% were grown in MCM in 48-well plates (100 μL) at 37 °C in the presence of heparin, CSA, HS, and dextran sulfate from 100 to 0.01 μg/mL for 48 h without changing medium. The concentrations of recombinant RON3HIS, SERA5-GST, MSP1-42-GST, MSP1-33-HIS, and GST 2187



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used in the inhibition test were 500, 250, 100, 50, and 10 μg/ mL, respectively. All samples were tested in triplicate three times. In order to quantify the parasitemia, the samples were stained with ethidium bromide (EB) and analyzed using an FACS (BD FACS Aria cell sorter, CA, USA). 50 000 cells were counted in each experiment.

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RESULTS AND DISCUSSION

Identification and Characterization of Heparin-Binding Proteins

Heparin and derivatives have been shown to inhibit erythrocyte invasion of P. falciparum parasites and to desequester infected red blood cells in the microvasculature.16,17 To investigate and understand the mechanism of heparin-mediated inhibition on erythrocyte invasion, we performed receptor-based affinity purification with lysates of P. falciparum schizont-infected erythrocytes using heparin-conjugated sepharose followed by high-throughput identification of the heparin-binding proteins. The reason for using heparin-sepharose to pull-down proteins from late stage schizonts instead of free merozoites is to avoid the potential loss of proteins loosely attached to the merozoite surface. Proteins that bound to heparin were first detected by Western-blot with human hyperimmune sera, and multiple polypeptides were detected to bind to heparin sepharose (Figure 1A−C). Further, the binding of the proteins to heparinsepharose beads could only be competed out by soluble heparin at relatively high concentrations (≈100−500 μg/mL; Figure 1B). CSA did not show any inhibitory effect on the binding of the proteins to heparin-sepharose (Figure 1C), and no binding was seen to unconjugated sepharose-beads suggesting the binding of a large number of the polypeptides to be specific for heparin. To identify and categorize the heparin-binding proteome of P. falciparum, the eluted proteins from the heparin-sepharose beads were further analyzed by multidimensional liquid chromatography tandem mass spectrometry (LC-LC-MS-MS) after trypsinization. The MS/MS spectra were searched against a combined database of all possible predicted tryptic peptides derived from P. falciparum, human and bovine (Supporting Information 2). An example of spectra of identified peptides was shown in 2. A total of 6062 peptides including fixed modifications mapped to 811 parasite proteins were identified (Supporting Information 3). More than 99% peptides were of P. falciparum origin with only 87 peptides matched to humanor bovine proteins (Table 1 and Supporting Information 2) suggesting the binding to heparin was specific. The proteins identified were categorized into 14 functional groups (Figure 3A) with various numbers in each group. Since proteins with high affinity to heparin would be more enriched, we therefore sorted the targeted proteins according to peptide

Figure 3. Categorization of heparin-binding proteins identified by proteomic analysis. (A) Functional classification of heparin-binding proteins identified by LC/LC−MS/MS. 811 unique P. falciaprumderived proteins enriched by affinity purification were identified by LC/LC−MS/MS. The proteins were categorized into 14 functional groups (some proteins can be categorized into several functional groups). The number of proteins in each group was labeled on each bar. (B) The most enriched proteins identified by LC/LC−MS/MS. Sixteen proteins with the most enriched peptides identified in the LC/ LC−MS/MS analysis were listed. They are functionally grouped into three categories: ligands for erythrocyte invasion, metabolic components, and unknown function.

counts (Figure 3B). Among the 16 proteins with more than 30 peptide counts per protein, 8 proteins belong to the pathogenesis group, and they are all associated with merozoite invasion or the parasitized erythrocyte membrane (Figure 3B and Supporting Information 3). Interestingly, members of the PfRhopH complex,28 RhopH2, RhopH3, and RhopH1 (also called CLAG3.2/CLAG3.1)28,29 and the Rhoptry neck protein 3 (RON3), are among the proteins with high affinity to heparin. The protein with the highest peptide count was RhopH2, a 140 kDa molecule retained in the rhoptry organelle and released upon the contact of the anterior end of a merozoite with the RBC surface during invasion. It is one of the critical molecules in the invasion process,30 while its erythrocytic receptor is still not known. RON3 is a molecule of the RON family which has been commonly identified in the phylum of Apicomplexa. However, unlike RON2 and 4, RON3

Table 1. Origin and Number of Proteins Identified in the Proteomic Analysis origins

number of proteins

P. falciprum-derived proteins trypsin (swine) bovine serum albumin bovine complement C3 precursor human cytoskeletal proteins human albumin hemoglobin

811 1 2 2 5 1 1 2188



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central domain which is a putative core of protease activity. The protein is synthesized in late trophozoite and schizont stage, which is released upon schizont rupture. But SERA5 is also attached to free merozoites. The primary role of SERA5 is believed to facilitate merozoite egress,40 but the finding of its interaction with heparin and erythrocyte membrane suggested that SERA5 may directly participate in the receptor engagement during merozoite invasion process. Four intracellular proteins dominantly enriched in the heparin-binding assay were P. falciparum 6-phophofructokinase (PFK), heat shock protein 90 (HSP 90), PfEMP2, karyopherin beta and methionine-tRNA ligase (Figure 3B). PFK is an ATPdependent enzyme participating in glycolysis.41 HSP 90 is a molecule composed of three subdomains with ATPase activity,42 which is a critical molecule for the parasite, not just because it is inducible in responses to stress, but also as a regulator in cell cycle, kinase activity, and transcription activation.43 PfEMP2, also called mature parasite-infected erythrocyte surface antigen (MESA), is a 250−300 kDa phosphorylated protein mainly localized under the membrane of the infected erythrocytes. It interacts with erythrocytic band 4.1 to stabilize cytoskeleton of the infected RBC.44 Karyopherin beta is a cytoplasmic protein with function as a nuclear transporter. It is immunogenic and antibodies to this protein have been shown to be protective.45 Methionine-tRNA ligase is an enzyme that catalyzes the formation of methionine and tRNA conjugate in the protein expression process. Only two proteins (MAL13P1.308, PF08_0091) (Figure 3B) without known function were captured with high heparin-binding activity in this study. Both seem to be conserved in parasites across different Plasmodium species and are expressed mainly in the late schizont stage. Though the significance of the intrinsic heparin-binding property of these intracellular proteins remains to be further studied, the majority of proteins identified in this study were erythrocyte invasion-associated molecules, which strongly suggests that P. falicparum may use heparin-like GAGs as receptors for erythrocyte invasion. It is known that microbial polypeptides with a heparin- or GAG-binding property contain clusters of positively charged amino acid residues in the consensus sequences XBBXBX or XBBBXXBX where B is a basic residue such as histidine, lysine, or arginie.27,46 Deep analysis of the polypeptides identified in this study found that all contained various numbers of potential GAG-binding motifs in the sequences (Supplemental Figure 1). And the recombinant protein domains tested for heparinbinding here all contain various number of heparin-binding motifs. However, it cannot be ruled out that the domains that were not successfully expressed also participated in the binding activity. Further, heparin-binding proteins in P. falciparum other than the ones identified here have been reported. For example, variants of P. falciparum erythrocyte membrane protein 1 (PfEMP1) were the first reported blood stage antigens with heparin-binding activity in association with the rosetting phenotype and sequestration of infected erythrocytes.13,16,47 However, heparin-binding variants of these proteins were not expressed by the 3D7 stain P. falciparum and proteins of PfEMP1 families are less soluble; they were therefore not identified in this study.

is mainly located in the rhoptry body and functionally interacts with RON2 and 4, but does not form complex with AMA1.31,32 Thus, heparin could inhibit or deassociate its interaction with RON2 and 4. The data further confirmed the finding that most of the rhopty proteins are in complexes, and one member of each complex may need to perform the main binding function to the host cell receptor. CLAG3.2 and CLAG3.1 are two paralogues of RhopH1 encoded by either PFC0110w or PFC0120w which are highly homologous to each other in sequence and possess similar secondary structures with other rhoptry family members, albeit with expression variation among different parasite isolates.28 The expression of the two paralogues is mutually exclusive;33 thus only one of the two paralogues should be identified in the study. The reason that peptides matching both CLAG3.2 and CLAG3.1 were identified in this study may be due to the heterogeneity of the 3D7 P. falciparum parasites which have been propagated in cultivation for many years. CLAG3.2 and 3.1 possess dual functions in both erythrocyte invasion and intracellular development. It is obvious that they participate in the junction formation between the merozoite and RBC membrane and the erythrocyte membrane indentation. However, they are also transported from the intracellular parasite and translocated to the membrane of infected RBC, to function as a channel for nutrient uptake.29 Thus, heparin may interfere with both functions of the RhopH1/CLAG3 molecules. Like RhopH2, RhopH3 is essential for the parasite, either in invasion or during intraerythrocytic development, since disruption of any of the genes will jeopardize the parasite proliferation. RhopH3 has been reported to be conserved among plasmodial parasites,34 and synthetic peptides of RhopH3 bound to erythrocytes and blocked merozoite invasion.35 Here we found that RhopH3 can be specifically precipitated by heparin, which suggests a role of this molecule in the invasion process. Rhoptry-associated protein 1 (RAP1) was also identified among the heparin-binding proteins. RAP1, 2, and 3 are low molecular proteins synthesized in the early trophozoite stage before the formation of rhoptries. They, through interaction with rhoptry-associated membrane antigens (RAMA),36 are retained in the bulb of the rhoptry organelles and the parasitophorous vacuole (PV)37 with a function in molecular sorting and trafficking. It was reported that RAP1 forms heterodimers with RAP2 or RAP3 in the rhoptries. Here only RAP1 was enriched through binding to the heparin beads, suggesting that only proteins with heparin-binding properties will be enriched. Taken together, the data illustrate that heparin can interact with a number of functionally critical proteins of the rhoptry complex. The consequence of such interaction can either be an inhibition of complex assembly or blockage of their binding to receptors on erythrocyte membrane in the sequential process of merozoite invasion. Apart from the rhoptry complex proteins, three proteins associated with the merozoite surface were also identified including MSP1, QF122, and serine repeat antigen 5 (Figure 3B). MSP1 has been previously reported to be a heparinbinding protein;17 here MSP1 was the second most enriched molecule on the heparin-sepharose, which further supports the finding that the major membrane protein of P. falciaprum merozoite interacts with heparin-like GAGs during erythrocyte invasion. QF122 antigen (PF10_0115) was found to be expressed in gametocytes, merozoites, and in the ring stage trophozoites.38 Serine repeat antigen 5 (SERA5) is a member of the papain family of cysteine proteases.39 It possesses a

Confirmation of Heparin-Binding and Invasion Inhibition with GAGs and Recombinant Proteins

Genes encoding the 16 proteins with prominent heparinbinding properties (Figure 3B) were cloned into expression 2189



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Table 2. Protein Fragments Tested for Heparin Binding serial no.

locus

protein name

1

PFI1445w

RhopH2

2

PFI1475w

MSP1

3

PFL2505c

RON3

4

MAL13P1.308

5

PFC0110w

conserved plasmodium protein-1 CLAG 3.2

6

PFC0120w

CLAG 3.1

7 8

PFB0340c PFI0265c

SERA-5 RhopH3

9 10

PF14_0102 PFI0755c

11

PF08_0091

12

PF07_0029

RAP1 6-phospho fructokinase conserved plasmodium protein-2 HSP90

13 14 15 16

PFE0040c PFE1195w PF10_0115 PF10_0340

PfEMP2 karyopherin beta QF122 antigen methionine-tRNA ligase

expressed regiona

expression vector

recombinant protein weight (kDa)

heparin-bindingb

1A:Leu -Arg 1B:Lys788-Tyr1373 2A: Val20-Glu724 2B:Thr725-Thr913 2C:Ser914-Thr1329 2D:Val1330-Leu1704 2E:Val1330-Leu1606 3A:Leu941-Arg1305 3B:Phe1372-Lys1779 3C:Glu1822-Pro2215

PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PET32a PGEX4T-1 PET32a PGEX4T-1

88.6 95.4 106.4 47.7 72.3 69.2 50.6 70.2 66.1 73.3

ND ND ND − − + + ND + ND

4A:Asp728-Lys1147 4B:Leu1148-ARG1514 5A:Asn49-Lys442 5B:Ala620-Gln1177 6A:Leu75-Glu512 6B:Ala618-Glu1177 Thr391-Asn828 8A:Leu36-Gly439 8B:Thr660-Pro865 Glu192-Asp782 10A:Asn56-Arg464 10B:Leu786-Arg1214 11A:Lys42-Met389 11B:Thr911-Glu756 11C:Asn872-Glu1210 12A:Met1-Lys206 12B:Val277-Asp745 Asp522-Lys873 Met616-Lys1057 Thr498-Asn940 Phe225-Leu579

PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1 PGEX4T-1

72.9 66.8 72.6 91.9 78 91.6 76.8 73.7 49.5 96.0 71.7 74.5 67.1 67.3 66.5 49.2 80.8 66.3 76.1 76.9 67.2

ND − + ND ND ND + ND + ND ND − − + + ND + ND ND ND ND

50

576

a

Protein fractions expressed. Numbers represent the start and end amino acids of each protein expressed. bND indicated the recombinant proteins were not in soluble form, and the test of heparin binding was unable to be performed. + indicated the binding to heparin was positive, and − indicated the binding to heparin was negative.

proteins identified here for their invasion blocking/inhibitory effects. We found that heparin, HS, and dextran sulfate inhibited merozoite invasion in a concentration-dependent manner, while CSA showed no effect on invasion (Figure 5A). In the presence of heparin and dextran sulfate at 100 μg/mL, the invasion rates were respectively reduced 61% and 73% compared to the control culture. HS had a weaker effect, showing an invasion inhibitory effect only at the concentration of 1000 μg/mL, with no effect at lower concentrations (Figure 5A). Previous studies suggested that the inhibitory effect of heparin was due to its interaction with MSP1, which blocks merozoite attachment to the erythrocyte surface.16,17 The data of this study suggest that heparin can interact with multiple merozoite-derived ligands. To further confirm the data, recombinant RON3, SERA5, MSP1-42, and MSP1-33 and a control protein (glutathione-Stransferase, GST) were tested for their invasion inhibitory effect. As illustrated in Figure 5B, RON3, SERA5, and MSP1-42 showed an invasion inhibitory effect in a concentrationdependent way, while MSP1-33 and GST did not. The difference between the effect of MSP1-42 and MSP1-33 confirmed that MSP1-19, which is missing in MSP1-33, is functionally critical in merozoite invasion. It has been previously reported that the C-terminal region of MSP-1

vectors, and the recombinant proteins were tested to obtain more evidence for heparin binding and to rule out the possibility that some of the purified molecules were carried over in protein complex during affinity purification. Most of the proteins were expressed as protein domains and not the whole molecules, since some were insoluble (Table 2). In total, 14 protein fractions from 16 molecules were successfully expressed (Supplemental Figure 2 and data not shown) and tested for heparin binding. Of the proteins tested, nine showed specific binding to heparin beads (Table 2, Figure 4A), and importantly, the binding can be competed out by heparin in a concentration-dependent manner (Figure 4B, Supplemental Figure 3). CSA did not show any inhibitory effect, indicating the binding was specific. Recombinant proteins of RhopH2, CLAG3.1, PfEMP2, karyopherin beta, QF122 antigen, and methionine-tRNA ligase were not soluble after expression; thus their binding property to heparin could not be tested. However, apart from the conserved P. falciparum protein (MAL13P1.308) and the 6-phospho fructokinase, regions of heparin binding of the soluble proteins have been identified (Table 2). Heparin and polypeptides of merozoite associated proteins have been demonstrated previously to inhibit P. falciparum merozoite invasion.16,17,32,48 Here, we tested heparin, HS, CSA, and dextran sulfate and four recombinant merozoite-associated 2190



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Figure 5. Merozoite invasion inhibition by heparin and heparinbinding proteins. Panel A shows the inhibitory effect of heparin and other GAGs on merozoite invasion of P. falciparum. Heparin, CSA, heparan sulfate, and dextran sulfate diluted in MCM with a concentration from 0.01 to 100 μg/mL were incubated with developmentally synchronized parasites for 48 h respectively. The inhibitory efficiency on invasion relative to the control (no GAG added) was calculated. Heparin and dextran sulfate showed a similar inhibitory effect at concentrations of 10 and 100 μg/mL, while heparan sulfate only had an inhibitory effect at 1000 μg/mL. No inhibitory effect with CSA was observed at any concentrations tested. Panel B shows the inhibitory effect of heparin-binding proteins. Similar to the invasion inhibition assay with the GAGs, recombinant RON3, SERA5, MAP1-42, MSP1-33, and GST were added to the culture with a concentration from 500 to 10 μg/mL. RON3, SERA5, and MSP1-42 showed a dose-dependent inhibitory effect, while MSP1-33 and GST do not have any effect on parasite invasion at the concentrations tested.

Figure 4. Heparin-binding of the recombinant merozite-associated proteins. Fragments of recombinant RON3, SERA5, MSP-1-42, and MSP-1-33 were tested for their heparin-binding activity. Panel A shows that all four proteins bound to heparin, no binding was seen to sepharose beads, and the GST tag did not bind to heparin. Panel B shows the inhibition of heparin on the binding of recombinant RON3 (①), SERA5 (②), MSP1-42 (③) and MSP1-33 (④) in a concentration from 0.001 to 100 mg/mL before mixing with heparin-sepharose. The proteins bound to heparin-sepharose were detected with specific antibodies that recognized HIS or GST tag. The binding of the four recombinant merozoite proteins to heparin-sepharose can be inhibited by soluble heparin in a concentration-dependent manner.

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interacts with the exofacial regions of human band 3 during merozoite invasion,49,50 and thus the inhibitory effect of recombinant MSP1-42 on merozoite invasion may be due to its interaction with both heparin-like glucosaminoglycan and band 3 receptors. The data collectively suggested that the merozoite-associated heparin-binding proteins are functionally important in erythrocyte invasion. In summary, we presented a new approach by combination of receptor-based purification and high-throughput proteomic identification of parasite proteins. Our data propose that heparin can interact with multiple merozoite-derived ligands and consequently has a profound inhibitory effect on red blood cell invasion compared to other GAGs. The data further support the exploration of heparin derivatives as antimalaria drugs. The work not only explains the mechanism of antiparasite invasion effect of heparin, but also suggests that the approach can be applicable to study host−pathogen interaction in general.

ASSOCIATED CONTENT

S Supporting Information *

Supplemental Figures 1−3. Supporting Information 1−3 (tables). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(Q.C.) E-mail: [email protected]. Tel/Fax: +86 431 87836701. (M.W.) E-mail: [email protected]. Tel: +46 8 524 800 00. Fax: +46 8430525. Author Contributions #

Y.Z. and N.J. contributed equally to the manuscript.

Notes

The authors declare no competing financial interest. 2191



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ACKNOWLEDGMENTS We very much appreciate the kind assistance of scientists at the BGI-Shenzhen for the bioinformatic analysis of the data. This study was supported by the National Natural Science Foundation of China (81130033, 81171592) and the National Science & Technology Specific Projects (2008ZX-10004-011).

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