Abstract
Intracellular Ca2+ mobilization induced by second messenger IP3 controls many cellular events in most of the eukaryotic groups. Despite the increasing evidence of IP3-induced Ca2+ in apicomplexan parasites like Plasmodium, responsible for malaria infection, no protein with potential function as an IP3-receptor has been identified. The use of bioinformatic analyses based on previously known sequences of IP3-receptor failed to identify potential IP3-receptor candidates in any Apicomplexa. In this work, we combine the biochemical approach of an IP3 affinity chromatography column with bioinformatic meta-analyses to identified potential vital membrane proteins that present binding with IP3 in Plasmodium falciparum. Our analyses reveal that PF3D7_0523000, a gene that codes a transport protein associated with multidrug resistance, as a potential target for IP3. This work provides a new insight for probing potential candidates for IP3-receptor in Apicomplexa.
Introduction
The inositol 1,4,5-triphosphate (IP3) is an important second messenger that regulates cytosolic Ca2+ in a variety of Eukaryotic organism(1, 2). Briefly, the activation of phospholipase C (PLC) mediated by surface receptor breaks phosphatidylinositol 4,5-bisphosphate (PIP2) into soluble short life second messenger IP3 that binds into IP3 receptor (IP3R) culminating in intracellular Ca2+ release(3, 4).
The phylum Apicomplexa includes unicellular eukaryotes parasites like Plasmodium, the etiology agent of malaria infection, and possesses the metabolic enzymes responsible for generation and degradation of IP3, see review (5). IP3 can mobilize Ca2+ from intracellular stores in isolate and permeabilize blood stage P. chabaudi(66) and in intact P. falciparum within red blood cells (RBCs). Within RBCs, parasites manage to maintain the Ca stores full even under low Ca2+ environment(8). An increasing number of reports supporting the existence of intracellular Ca2+ release induced by IP3 in malaria parasites(6, 7, 9–12) suggesting the existence of a Ca2+ channel sensitive to IP3, the IP3R.
The IP3R it is a well know described protein in vertebrates that contains around four to six transmembrane domains (TMDs), see review(13). Prole and Taylor(14) used the sequence of mammal N-terminal IP3R binding domain and the amino-terminal RIH (Ryanodine and IP3R homology) domains to perform a BLAST (Basic Local Alignment Search Tool) on the genome of diverse parasites. However, this work failed to find any potential candidate for IP3R in Apicomplexa. So far, no apicomplexan IP3R candidate has been identified through bioinformatics approaches. Moreover, there is no publication that attempted to use biochemical approach like an IP3 affinity chromatography column in Apicomplexa to identify proteins that might bind to IP3.
Hirata and collaborators(15) managed to enrich proteins from rat brain sample that has an affinity to IP3 like IP5-phosphatase and IP3 3-kinase using an analogous IP3 affinity chromatography column 2-O-[4-(5-aminoethul-2-hydroxyphenylazo) benzoyl]−1,4,5-tri-O-phosphono-myo-inositol trisodium salt-Sepharose 4B. Using a similar column, Kishigami and collaborators(16) managed to identify the PLC protein from octopus’ eyes Todarodes pacificus and reported that squid rhodopsin also has an affinity to IP3. Nevertheless, besides the potential of these columns to enrich proteins that bind to IP3, no IP3R has ever been identified using a IP3-affinity column alone.
By adapting the protocol from Hirata/Kishigami(15, 16), we created a column containing IP3 conjugated with biotin linked with a high-performance sepharose-streptavidin and challenged with proteins from asynchronous P. falciparum blood-stage. Using a IP3-free column containing only sepharose-streptavidin as reference, we selected only the candidates exclusive on the IP3-column to undergo a series of bioinformatic meta-analyses. Our approach targeted for candidates with at least one transmembrane domain, considered essential, conserved among most apicomplexan species, and with unknown or nor-clear function. Finally, the candidate that fit all these criteria were used as targets for in silico molecular docking against IP3.
Using this strategy, we identified the P. falciparum multidrug resistance protein 1 (PfMDR1), a vital and conserved membrane protein that has potential to bind to IP3. This protein is located on parasite food vacuole, a Ca2+ storage compartment(17). Combined, the affinity column and bioinformatic successfully provided a candidate for a membrane protein associated to an intracellular Ca2+ compartment that binds to IP3 in malaria.
Material and Methods
P. falciparum Culture
P. falciparum (D37) parasites were maintained in culture as described(18). Briefly, P. falciparum were cultured in RPMI media supplemented with 50 mg/L hypoxanthine; 40 mg/L gentamycin; 435 mg/L NaHCO3; 2% haematocrit of A+ human red blood cells and 10% A+ human blood serum in an atmosphere of 5% CO2; 3% O2; 92% N2 at 37°C. Media was changed every 24 h and RBCs replaced every 48 h. Parasitemia and the development stage of cultures were determined by Giemsa-stained smears.
P. falciparum protein sample
Total P. falciparum protein extract was obtained from 2.5 L of unsynchronized culture, at 8% parasitemia. Culture was washed three times in PBS (300 g, 5 minutes) and parasites isolated from erythrocytes using 0.03% (w/v) saponin (Sigma) on PBS containing protease inhibitors: antiplaque, pepstatin, chymostatin and leupeptin (Sigma) at concentrations of 20 μg/mL each and 500 μM benzamidine (Sigma). Isolate parasites were centrifuge on 1300 g for 10 minutes at 4°C and washed three times in PBS with protease inhibitors. The isolate parasite samples were resuspended in 50 mM TRIS-HCl buffer pH 7.4 containing 2 mM EDTA, 0.1% Triton X−100, protease inhibitors and 1 mM PMSF. Samples were sonicated on SONIC (Vibracell) 50% potency for 20 seconds for 3 times on ice (10-second interval between each sonication) follow by at 1300 g centrifugation for 10 minutes at 4°C for removal of the insoluble pellet. DNAse and RNAse (final concentration 200 ng/μL each) were added on soluble pellet and incubate for one hour at 37 °C. The samples were passed through a 0.45 μm filter. The amount of protein was quantitated using Pierce’s BCA protein assay kit.
IP3-affïnity chromatography column
To build the column, it was used a commercial high performance Sepharose substrate bound to streptavidin (GE Healthacare Life Science) and biotin-conjugated IP3 (Echelom Biosciences). The streptavidin-sepharose column was equilibrated by washing once with 10x volume of ice-cold, 0.45 μm filter binding buffer (20 mM NaH2PO4, 150 mM NaCl, 20 mM LiCl and 2 mM EDTA, pH 7.5). The columns were mounted in a 15 mL sterile falcon tube. For each column it was used 1.25 ml of equilibrated S epharos e-streptavidin resuspended in binding buffer mixed with 20 μg of IP3-biotin. The columns were left by constant stirring for 12 hours at 4°C in a dark environment and then centrifuged for 1 minute, 300 g at 4 °C. The supernatant containing excess IP3-biotin was removed and columns were washed five times with 2 ml of ice-cold binding buffer to remove any free IP3-biotin. Two distinct columns were assembled: one containing IP3-biotin-sepharose-streptavidin and other containing sepharose-streptavidin only. In each column was added 2.5 mg of P. falciparum protein extract. The column volume was adjusted with ice-cold binding buffer with protease inhibitors until a final volume of 5 ml. The columns were incubated at 4°C under gentle, steady shaking in a light-protected environment for 12 hours. After incubation, the columns were centrifuged for 1 minute at 300 g at 4 °C and the supernatant discarded. Each column was washed seven times with ice-bound binding buffer with protease inhibitors. To elute the proteins, 1mL of an ice-cold elution buffer (8 M Guanidin-HCl, 20 mM LiCl, 2 mM EDTA, pH: 1.5 with protease inhibitors) was added on each column and followed by constant stirring for 1 hour at 4 °C. At the end of this incubation the columns were centrifuged for 1 minute at 300 g and the supernatant collected in sterile low binding protein eppendorfs.
Mass spectrometry
The protein samples were applied on 8% polyacrylamide gel and run at low voltage (60 v) until the bands were discriminated. After the run, the gel was fixed and stained following the recommendations of the “Colloidal Blue Staining Kit” from Invitrogen. The sections of the gel containing visible bands were cut and sent for analysis on a mass spectrometer at Taplin Mass Spectrometry, Harvard Medical School (https://taplin.med.harvard.edu/) for protein identification. All identified proteins containing at least one exclusive peptide match was considered for analyses.
Transmembrane domain prediction
To detect the presence of a transmembrane domain, the whole amino acid sequence from the protein identified at mass spectrometry were analysed using the public HMMTOP program version 2.0 (www.enzim.hu/hmmtop/). This program predicts the number of transmembrane helicases and their position from peptide/protein amino acid sequence.
Phenotype score, conservation and function predictions
The phenotype score used the determinate gene essentiality of each candidate was obtained from the work of Zhang and collaborators(19) that is available on PlasmoDB (https://plasmodb.org/plasmo). To identify the presence of a candidate orthologs among Apicomplexa group, we use the OrthoMCL database (https://orthomcl.org/orthomcl). For function prediction, we consulted the gene annotation information provided by PlasmoDB.
In silico docking with IP3
The primary sequence of MDR1 (Gene - PF3D7_0523000, plasmodb.org) was used to build its probable 3D structure by homology modelling. The server SwissModel(20) was employed to automatically build the models optimized to bind IP3 at various locations inside MDR1 homology. Blind molecular docking simulation were carried out to obtain possible interactions for the intermembrane domain as predicted by the TMHMM Server(21). The SwissDock(22) server enabled the study of IP3 intermembrane MDR1 domain binding poses. Additionally, the IP3-Ion-MDR1 binding was further investigated using the multidrug transporter permeability (P)-glycoprotein is an adenosine triphosphate (ATP)-binding cassette (PDB id: 6C0V). The later ability to bind simultaneously ATP and a divalent cation at the intracellular domain was used to guide the inspect a hypothetical IP3-Ion-MDR1 interaction. IP3 was manually positioned inside the ATP cavity to mimic an IP3-Mg2+ interaction. The binding conformation was optimized with molecular mechanics by means of the UCSF(23) chimera minimize structure tools.
Results
IP3-affinity chromatography data
Adapting the protocol based on Hirata/Mishigami(15, 16), we use an IP3 affinity chromatography column with protein homogenate from unsynchronized asexual blood stages of isolated P. falciparum as the first step to identified potential proteins that have a similar function to IP3R receptor in a mammal.
The access code of the brute data on mass spectrometry analyses from the eluate samples of IP3-affinity chromatography column can be found in Supplemental Material Table 1. At least 700 proteins from P. falciparum containing at least one exclusive peptide were detected from the IP3-sepharose column. In comparison, 494 proteins were detected from the sepharose matrix alone (Figure 1). With both columns’ information, all proteins exclusively present on IP3-sepharose were selected (total 206 proteins) for the bioinformatic meta-analyses (Sup. Table 2).
Once the proteins exclusive for IP3-column were identified (Sup. Table 2), the first bioinformatic approach aimed to select proteins that contains a transmembrane domain (TMD). The IP3R is a protein attached at the membrane, a TMD is an important structure to anchor proteins through biological membranes by its physical properties like the length and hydrophilicity of the transmembrane span(26), every IP3R in vertebrates, invertebrates and single eukaryotes organism possess a TMDs, so we use this feature as the second step to select potential candidates for IP3R. Figure 1.
The table 1 summarizes the list of 26 proteins exclusively found at IP3-biotin-streptavidin-sepharose column that contains at least one TMDs. Transfection of P. falciparum to constitutively express IP3-sponge, a protein containing a modified IP3 binding domain based on mouse IP3R that sequestrate cytosolic IP3(27), does not result in viable parasites(28) suggesting a vital role of IP3 signalling in P. falciparum. With this information, the next step to narrow the number of potential candidates that might act as IP3R in malaria is focus on essential genes. To deem whether a gene is essential, we considered only the genes that score lower than 0.5 on its mutagenic index of phenotype graphic (data provide by PlasmoDB). That decrease the number of candidates to 11 (Fig. 1, table I).
For the next step on the bioinformatic meta-analyses, we considered only the genes that is conserve among multiples species within Apicomplexa phylum. Only four essential candidates with TMDs domains met this criterium: multidrug resistance protein 1 (MDR1); a heat shock protein 40, type II (HSP40); aspartate carbamoyltransferase (ATCase) and antigen UB05. PlasmoDB access code: PF3D7_0523000, PF3D7_0501100, PF3D7_1344800 and PF3D7_1038000 respectively. Among these 4 candidates, only MDR1 and antigen UB05 has unknow or unclear function. The HSP40 is a cochaperone protein with conserved J-domain that regulates other heat socks protein 70 (HSP70)(29) and the ATCase is an enzyme important for the pyrimidine biosynthetic pathway(30).
IP3-MRD1 binding modelling and protein interactions network
The MDR1 model provide by the SwissModel server proved to be quite similar to human P-glycoprotein ABCB1 receptor, protein data bank id: 7A69(31). The sequence alignment proved that a homology model could be built with fair quality with an identity of 29.7% and similarity of 48.2% (Pairwise Sequence Alignment EMBOSS Water server, https://www.ebi.ac.uk/Tools/emboss/). Two binding position at the transmembrane domain of MRD1 and IP3 binding was estimated by the SwissDock server (Figure 2).
The pocket 1 (binding energy −15.7 kcal/mol) proved to be the best IP3 docking position. The site is a lysin rich domain able to form various hydrogen bonds with IP3. The second-best bind pocket proved to be less favored as derived from the lower interaction energy (−11.4 kcal/mol). Another binding possibility investigated was the interaction with the same pocket ATP binding. The interaction involves the presence of a divalent cation (green spheres) like Mg2+ intercalating with IP3. The MDR1 is an ATP-binding cassette (ABC) transporter family member that is associated to multidrug drug resistance due the ability to translocating amphiphilic compounds(32). The translocation of a substrate across the membrane by proteins like P. falciparum MDR1 requires an ATP binding on Q-loop site that cause a rearrangement of TM(33). The binding on IP3-divalent cation on the MDR1 Q-loop site suggests a potential competition between ATP and IP3. Interestingly, ATP is known to allosterically modulate the functional of mammal IP3R(34, 35) including the inhibition of Ca2+ flux regulated by IP3R under high concentration of ATP(35).
To help uncover the cellular function of MDR1 protein, we searched for proteins that interact with MDR1 in the Plasmodium interactome data(24) (Figure 3). It is interesting to note that MDR1 interacts with receptor for activated C kinase (RACK1, PF3D7_1148000). The PfRACK1 can inhibit host IP3-mediated Ca2+ signaling by direct interaction with IP3R(36). This data suggests that PfMDR1 has the potential to associate or have similar functions to other receptors that coordinate signaling events regulated by protein kinase C.
Discussion and conclusion
Phylogenetic analyses and comparative genomic managed to reveal both unique and conserved proteins related to calcium signalling pathways on apicomplexan parasites(14, 37, 38), nevertheless the IP3R still remain a major missing piece of this Ca2+ signalling toolkit. The conflict with the pharmacological and functional studies using exogenous IP3 that supports the existence of protein sensitive to IP3 that is capable to mobilize Ca2+ with the bioinformatic data that constantly failed to point an IP3R candidate suggests this receptor in Apicomplexa has a different and unique structure compared to the IP3-binding core domain from other eukaryotes. The search for an IP3R in Apicomplexa requires a different strategy that do not rely exclusively on bioinformatic tools as BLAST (Basic Local Alignment Search Tool) based on previously known IP3R.
The use of IP3 affinity chromatography column has being successfully reported to concentrate proteins that interact with high affinity to IP3 analogues(15) as well as retained key components from IP3-Ca2+ signalling from proteins extract from tissues(16). In this work, we used a biotin-inositol 1,4,5-triphosphate attached to a high-performance streptavidin-sepharose substrate to initially enrich proteins with IP3 affinity from unsynchronized isolate P. falciparum blood culture. One of the major limitations of using chromatography affinity column based on a short life IP3 molecule is the number of proteins that are naturally present at sample homogenate that degrades this second messenger. P. falciparum contains proteins that can dephosphorylate or phosphorylate IP3 like inositolpolyphosphate 5-phosphatase and inositol 1,4,5-trisphosphate 3-kinase(39). In this protocol, we tried to overcome this limitation by keeping all the binding and elution steps under low temperature while adding LiCl in every buffer. LiCl has being previously used to inhibit the dephosphorylation of IP3 (40–42). Another risk of using an IP3 affinity column is assume that protein(s) that might act as IP3R in Plasmodium do not bind/interact with strong affinity with the sepharose-streptavidin substrate alone. In this protocol, we excluded all 494 proteins that bind with the sepharose-free IP3 column as a potential IP3R candidate (Fig.1).
From the 206 proteins identified exclusively from IP3-sepharose column, 180 did not contained any TMDs suggesting the protocol used to extract the proteins from parasite lysate benefit mostly soluble proteins that do not strongly interact with lipid bilayers. For future trials, this protocol can be strongly optimized by using a protein extraction that target membrane proteins (MPs). The IP3R in mammals is a MP protein containing 6 TMDs(43). The presence of a TMDs is an important aspect of any MPs to physically interact with biological membranes(26). It is fair to predict that any protein with potential function of IP3R should have TMDs to able to interact with membranes. The table I list all candidates with TMDs exclusively from IP3-column.
Ca2+ is a second messenger that regulate a variety of vital functions in apicomplexan parasites(37, 44, 45). Accordingly, the use of 2-aminoethoxydiphenyl borinate (2-APB), a pharmacological drug that inhibit IP3R, abolished spontaneous Ca2+ mobilization and compromise intracellular development of blood stage P. falciparum(11. Pecenin and collaborator(28) failed to obtain any viable parasite expressing IP3-sponge. These data suggest that IP3-Ca2+ signalling pathway has a vital role during intraerythrocytic development of P. falciparum and support our hypotheses that a potential candidate for IP3R in Plasmodium not only has to present a TMDs, but also has to be essential. A prediction of gene essentiality in P. falciparum, based on the work of Zhang and collaborators(19) is available for consultation at PlasmoDB website.
The pharmacological evidence that supports IP3-Ca2+ signalling pathway in Apicomplexa group is not exclusive to malaria parasites, but also present in Toxoplasma gondii46’48 and Babesia bovis(49). The strategy to pinpoint the potential candidate for IP3R in apicomplexan should not rely on gene only excusive to Plasmodium species. Adding this extra meta-analysis step, the list of potential candidates presented exclusively on IP3-sepharose column is finally reduced to four proteins: a MDR1; a HSP40); an ATCase and antigen UB05. Among those four, only MDR1 and antigen UB05 has an unclear function.
The small number of candidates makes the use of more computational demanding bioinformatic analyses more feasible. A molecular docking allows to target the structural protein complexes from our candidate list against potential ligand as IP3 or other potent IP3-analogues drugs like adenophostin A(50).
Molecular docking on IP3 on P. falciparum MDR1 protein revealed two potential binding sites on TMD: pocket site 1 (binding energy −15.7 kcal/mol) and pocket site 2 (−11.4 kcal/mol), see Figure 2. This data suggests that MDR1 pocket 1 has a higher affinity to IP3 compared to IP3-binding core of mammal IP3R (ΔG= −10.3 kcal/mol on 23°C)(51) and a lower affinity when compare to IP3-binding with N-terminal region of mammal IP3R (ΔG= −79.5 kcal/mol)(52). Nevertheless, the binding of ATP on Q-Loop site on nucleotide-binding domain (NBD) likely cause profound changes on the TMD region(33)making hard to predict the real affinity of MDR1 protein with IP3.
In P. falciparum the MDR1 gene encodes for a 162.2 kilo Daltons P-glycoprotein located on the digestive vacuole (DV)(53) with unclear function, but the polymorphisms within this protein are associated to increase in vitro resistance against multiple antimalarial drugs like quinine(54–61). The MDR1 display a role as a transporter protein bringing solutes into DV and it is consisted of two distinct homologous regions: one cytosolic nucleotide-binding domain (NBD) and a substrate-binding consisting of 11 TMDs(62, 63). Interestingly, in malaria parasites the DV is an acid compartment known to be a dynamic intracellular Ca2+ store(17, 64–66), making the subcellular location of MDR1 protein suitable for a IP3R-like candidate. Moreover, the in vivo and in vitro treatment with IP3R inhibitor 2-aminoethoxydiphenyl borinate (2-APB) is associated to reverse resistance to antimalarial chloroquine in P. falciparum and P. chabaudi parasites, presumably by disrupting Ca2+ homeostasis(67). Multiple antimalarial drugs can also disrupt Ca2+ dynamic on parasite(68, 69), nevertheless there is not direct evident that suggest the MDR1 acts as a Ca2+ gate.
Considering that agents that disrupt IP3R channels such as 2-APB block malaria in vitro growth(9, 11, 28), identified this receptor in Plasmodium will not only add crucial missing information on malaria Ca2+ signalling but it will also present a potential new target for pharmacological treatment.
This work aims to stimulate the use of IP3-affinity column with bioinformatic strategies as a potential tool to identify proteins that might act as IP3R in Apicomplexa. The MDR1 seems to be a promising candidate to be validated, however, it is initial step from a long rewarding task of finding the Apicomplexa channel sensitive to IP3.
Conflict of Interest
The authors declare no conflict of financial or commercial interests.
Funding
Celia R. S. Garcia is funded by FAPESP (2017/08684-7; 2018/07177-7). Helder Nakaya is funded by FAPESP 2018/14933-2.
Acknowledgements
We thank Prof. Dr Akio Kishigami for the helpful suggestion on the IP3-Affinity Column. Ross Tomaino from Taplin Mass Spectrometry Facility for helpful support on mass spectrometry data.