Background
In spite of extensive control efforts, malaria continues to be a major health problem worldwide, causing approximately 438,000 deaths in 2015. In Africa alone, the death toll reached 292,000 among children under five years old [
1]. The malignant tertian parasite
Plasmodium falciparum accounts for the majority of fatal malaria infections. Severe pathologies such as organ failure and dysfunction, cerebral malaria, and placental malaria are most often associated with sequestration of the infected red blood cells (iRBCs) into the deep microcapillaries of these organs by adhering to endothelial cells. Cytoadherence is mediated by parasite proteins exported to the iRBC membrane. These proteins are first transported across the parasite plasma membrane and the parasitophorous vacuole membrane (PVM). Then they are sorted and translocated through the Maurer’s clefts and finally inserted into the iRBC membrane [
2,
3]. Maurer’s clefts are membranous structures involved in sorting and translocating parasite proteins to the iRBC membrane [
4,
5]. These extensive modifications of the iRBC dramatically alter its morphology, antigenicity and functions, including the appearance of knob protrusions on the iRBC surface, increased rigidity and poor deformability of iRBC, and increased adhesiveness of the iRBC to the endothelium [
4,
6,
7]. Some exported proteins such as PfEMP1, PfEMP3, MESA, Pf332, PfSBP1, KAHRP1, and RESA interact with RBC membrane skeleton [
7‐
13]. In addition, PfEMP1 family proteins encoded by the
var gene family bind to host factors such as CD36, ICAM-I, and CSA, mediating cytoadherence of the iRBCs and leading to severe pathologies.
Among
P. falciparum proteins that contain tryptophan-rich residues are the SURFIN family proteins. SURFIN
4.2 is one of the iRBC-exported proteins and is encoded by a small family of surface-associated interspersed (
surf) genes consisting of 10 members in the
P. falciparum genome [
14].
Plasmodium falciparum SURFINs form one clade with the
Plasmodium vivax subtelomeric transmembrane proteins (PvSTPs) [
15]. The intracellular tryptophan-rich domains (WRDs) of SURFIN/PvSTP are related to the sequences of the intracellular regions of PfEMP1 and Pf332 [
14]. SURFIN
4.2 localizes to Maurer’s clefts and has been reported to be trafficked to the surface of the iRBC together with RIFIN and PfEMP1 [
14]. Thus, SURFIN/PvSTP proteins are potential immune targets and malaria vaccine candidates [
16,
17]. For another member SURFIN
4.1, the N-terminal 50 amino acids, transmembrane domain, and adjacent intracellular region contain sufficient information for recruiting a recombinant protein into the classical ER/Golgi secretory pathway, and for efficient translocation across the PVM to the Maurer’s clefts [
18]. The mechanism by which SURFIN proteins are anchored into the iRBC membrane has yet to be elucidated, but recombinant SURFIN
4.2 possessing the intracellular WRD can be cleaved by surface treatment of iRBC with proteinase K, suggesting the WRD of SURFIN
4.2 may be responsible for transport of the protein from Maurer’s clefts to the iRBC membrane [
19]. Interestingly, intracellular region of Pf332 that is homologous to the SURFIN WRD is found to associate with actin filaments of RBC membrane skeleton [
12]. In the case of PfEMP1, the intracellular VARC region (also known as the acidic terminal sequence, ATS) having homology with WRD binds to host spectrin-actin [
4,
20,
21]. Thus, this study aimed to identify host RBC proteins that may associate with SURFIN
4.2 WRD. This could provide an important insight into the molecular basis of trafficking of SURFIN proteins from Maurer’s cleft to iRBC surface. This study revealed binding of WRDs of SURFIN
4.2 and PvSTP2 to RBC membrane skeleton proteins, and interactions between the second WRD of SURFIN
4.2 with actin and spectrin.
Discussion
The human RBCs are enucleated, terminally differentiated cells, and packed with hemoglobin that is responsible for oxygen and carbon dioxide transportation in the circulatory system. RBCs contain a membrane bound skeleton network primarily comprised of spectrin, actin, and protein 4.1, which make the RBCs highly deformable to pass through the capillaries/reticuloendothelial system without fragmentation. However, after malaria parasite invasion, the morphology and functions of iRBCs are dramatically modified, a process that contributes to the disease pathology. Malaria parasites as ‘master renovators’ of their host cells achieve these modifications by exporting hundreds of proteins into the iRBC cytosol [
27]. Some of these exported proteins are located to the surface of iRBC, such as the cytoadhesins PfEMP1, STEVOR, and RIFIN, and others including RESA, MESA, and PfEMP3, which become associated with the RBC membrane skeleton [
7,
10,
13,
28‐
31].
Furthermore, some proteins reside in the Maurer’s cleft, which acts as a sorting depot for proteins
en route to the surface of iRBC, including SBP1, REX1, and MAHRP1, which are responsible for PfEMP1 to be inserted into the plasma membrane of iRBC [
10,
32,
33]. Extensive studies in protein trafficking have identified the
Plasmodium exported element (PEXEL) in numerous parasite exported proteins. However, an even probably larger group of
Plasmodium parasites also exported is PEXEL-negative proteins (PNEPs), including PfEMP1, SURFIN family, and some Maurer’s cleft dotting proteins, which typically contain an internal transmembrane domain that functions as an ER sorting signal, and an essential N-terminal signal responsible for further localization to the iRBC surface or remaining in the cytosol to help remodeling the iRBCs [
18,
34‐
37]. The trafficking mechanism of most of the PNEPs including SURFIN is not fully understood.
As one of the variant surface antigens (VSAs),
surfin/pvstp gene family possesses 10 members in
P. falciparum and two members in
P. vivax; some were also detected in the genomes of
Plasmodium ovale curtisi and
Plasmodium ovale wallikeri [
14,
16,
38,
39]. Furthermore, previous hierarchical clustering analysis identified two
surfin/pvstp genes in
Plasmodium gallinaceum (
PgSurf1 and
PgSurf2), indicating that SURFINs are also conserved outside the human malaria parasites [
40]. Multiple sequence alignment revealed that SURFIN/PvSTP proteins,
P. falciparum PfEMP1 and Pf332, and
Plasmodium knowlesi SICA
var are interrelated through a modular and structurally conserved intracellular WRD [
14,
40]. PfEMP1 WRD binds to host spectrin-actin with high affinity (
K
d = 0.04 µM), and also interacts electrostatically with four linear sequence motifs in KAHRP to form a fuzzy complex and to govern the phenomena of knob formation and cytoadherence of iRBC [
4,
20,
21]. Furthermore, a 260-residue sequence within the WRD of Pf332 specifically interacts with F-actin (
K
d = 0.60 μM, [
12]). In the current study, WRD2 shared the most similarity between two known exported SURFINs, SURFIN
4.1 and SURFIN
4.2. Previous data have shown that recombinant SURFIN
4.2 containing WRDs were mainly detected in Triton X-100 insoluble fractions, compared to the one without WRD, and also exhibited a unique localization pattern in the iRBC cytosol, implying a direct interaction of these SURFIN
4.2 with the RBC membrane [
6]. Consistently, by fusing the conserved WRD2 of SURFIN
4.2 with the minimum Maurer’s cleft targeting motifs present in SURFIN
4.1 [
18], the recombinant NTC-4.2WRD2 also was targeted to the iRBC cytosol. Sequence conservation among WRDs suggested that WRD serves as a domain responsible for the binding to RBC membrane skeleton in most SURFIN/PvSTP proteins. To test this, in vitro binding experiments were performed by using other WRDs from SURFIN
4.2 and PvSTP2 proteins with IOVs. In almost all cases, direct SURFIN/PvSTP WRD binding was observed with
K
d values ranging from 0.26 to 0.68 µM. These modest binding values are of similar magnitude to the affinities of those observed in other
Plasmodium proteins that are associated with RBC membrane skeleton, such as Pf332 (
K
d = 0.40 µM) [
7,
12,
29].
The potential binding between WRD2 of SURFIN
4.2 and actin (an RBC membrane component) is implied from several clues. A previous report identified a fragment of 260 residues within the WRD of Pf332, which can bind to F-actin in a specific and saturable manner [
12]. Pairwise sequence alignment revealed high sequence similarity between Pf332 and SURFIN/PvSTP proteins in this region [
40], which makes actin the strongest candidate that interact with WRD2 of SURFIN
4.2. This study tested the direct interaction between WRD2 of SURFIN
4.2 and F-actin, which indeed confirmed such an interaction, albeit the interaction was relatively weak (
K
d = 5.16 μM), and this interaction was abolished by further truncation of the recombinant protein. This value was more than eight folds higher than that detected for Pf332 in the previous study (
K
d = 0.6 μM) [
12] and other known parasite-host interacting proteins, including PfCor-N (
K
d = 0.96 μM) and PfAldolase (
K
d = 0.37 μM) [
32,
41]. It is noteworthy that the
K
d values of all examined WRDs of SURFIN
4.2 and PvSTP2 with IOVs are similar and significantly higher than that of WRD of Pf332 and IOVs. Thus, the
K
d value for the WRD2 of SURFIN
4.2–F-actin interaction likely reflects the nature of the WRD2 of SURFIN
4.2. It is tempting to speculate that the weak affinity of
P. falciparum SURFIN WRD may be the driving force of the multiplication of this domain in the SURFIN family proteins in
P. falciparum, which need to compete with other
P. falciparum-specific proteins that also interact with actin and/or spectrin, such as PfEMP1, Pf332, and PfEMP3. However, it is also possible that the adjacent residues of the examined WRDs could enhance protein–protein interaction with RBC membrane skeleton, as has been reported for MESA interaction with protein 4.1 [
42]. These possibilities need to be evaluated in the future. Interestingly, this study also identified interactions between the CRD of SURFIN
4.2 and F-actin. However, this interaction may not be physiological since CRD and host F-actin are predicted to be located in different cellular compartments.
Another major component of the RBC membrane skeleton is spectrin, which is a flexible rod-like protein that predominantly exists as an α
2β
2 tetramer, and interacts with actin, protein 4.1, and ankyrin to form a network [
43]. Several trafficked proteins to the RBC membrane interact with spectrin. Among them, RESA binds to the β chain of spectrin with a 108 amino acid fragment (residues 663–770), and stabilizes the spectrin tetramer and enhances resistance of the iRBCs to both mechanical and thermal degradation [
29]. The spectrin-binding domain in KAHRP has been localized to a 72-residue region (residues 370–441), which is critical in membrane localization of KAHRP [
24]. In the current study, the WRD2 of SURFIN
4.2 also was found to interact with spectrin with the
K
d value of 0.51 μM, which is comparable to that of RESA (0.88 μM) and PfEMP3 (0.38 μM) [
29,
31]. Since no conserved motifs have been identified between SURFIN/PvSTP WRD and these previously identified spectrin-binding parasite proteins, it is possible that binding sites of spectrin for these parasite proteins might be different. The binding motifs in both spectrin and WRD2 of SURFIN
4.2, and the functional role of this binding to spectrin remain to be further evaluated. Interestingly, the data presented here showed that the WRD2 of SURFIN
4.2 was able to interact with both actin and spectrin. This is not an exception; a 14-residue fragment of PfEMP3 also has dual binding abilities to both spectrin and actin [
7]. This dual binding ability detected in SURFIN
4.2 and PfEMP3 may increase the affinity of these proteins for the iRBC membrane skeleton.