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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Malaria Journal 1/2017

Tryptophan-rich domains of Plasmodium falciparum SURFIN4.2 and Plasmodium vivax PvSTP2 interact with membrane skeleton of red blood cell

Malaria Journal > Ausgabe 1/2017
Xiaotong Zhu, Yang He, Yifan Liang, Osamu Kaneko, Liwang Cui, Yaming Cao
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12936-017-1772-5) contains supplementary material, which is available to authorized users.



Plasmodium falciparum dramatically alters the morphology and properties of the infected red blood cells (iRBCs). A large group of exported proteins participate in these parasite-host interactions occurring at the iRBC membrane skeleton. SURFIN4.2 is one of iRBC surface protein that belongs to surface-associated interspersed protein (SURFIN) family. Although the intracellular tryptophan-rich domain (WRD) was proposed to be important for the translocation of SURFINs from Maurer’s clefts to iRBC surface, the molecular basis of this observation has yet to be defined. The WRDs of P. falciparum SURFIN proteins and their orthologous Plasmodium vivax subtelomeric transmembrane proteins (PvSTPs) show homology to the intracellular regions of PfEMP1 and Pf332, both of which are involved in RBC membrane skeleton interactions, and contribute to malaria pathology.


Two transfected lines expressing recombinant SURFINs (NTC-GFP and NTC-4.2WRD2-GFP) of the 3D7 sequence were generated by transfection in P. falciparum. In vitro binding assays were performed by using recombinant WRDs of SURFIN4.2/PvSTP2 and inside-out vesicles (IOVs). The interactions between the recombinant WRDs of SURFIN4.2/PvSTP2 with actin and spectrin were evaluated by the actin spin down assay and an enzyme-linked immunosorbent assay based binding assays, respectively.


The recombinant SURFINs (NTC-4.2WRD2-GFP), in which the second WRD from SURFIN4.2 was added back to NTC-GFP, show diffused pattern of fluorescence in the iRBC cytosol. Furthermore, WRDs of SURFIN4.2/PvSTP2 were found to directly interact with the IOVs of RBC, with binding affinities ranging from 0.26 to 0.68 μM, values that are comparable to other reported parasite proteins that bind to the RBC membrane skeleton. Further experiments revealed that the second WRD of SURFIN4.2 bound to F-actin (K d = 5.16 μM) and spectrin (K d = 0.51 μM).


Because PfEMP1 and Pf332 also bind to actin and/or spectrin, the authors propose that the interaction between WRD and RBC membrane skeleton might be a common feature of WRD-containing proteins and may be important for the translocation of these proteins from Maurer’s clefts to the iRBC surface. The findings suggest a conserved mechanism of host-parasite interactions and targeting this interaction may disrupt the iRBC surface exposure of Plasmodium virulence-related proteins.
Additional file 1: Table S1. Primers for PCR amplification and plasmid construction.
Additional file 2: Figure S1. Sequence information of the His-tagged unrelated protein. The thioredoxin coding frame and protein sequences are shown in brown colour; The 6×His tag coding region and protein sequences are shown in blue colour; The multiple cloning sites are marked by underlines.
Additional file 3: Figure S2. Alignment of the tryptophan-rich domains (WRDs) of Pf332, SURFIN 4.2/PvSTP2, and PfEMP1. Included in analysis are Pf332 WRD amino acid positions 5568-5825, WRDs of SURFIN 4.2/PvSTP2 (WRD1 959-1201; WRD2 1349-1567; WRD3 1729-1990; PvSTP2 WRD 592-825), and PfEMP1 WRDs (PFD1005c WRD 1844-2181; PFD1015c WRD 1851-2193; PFL0030c WRD 2753-3056). Asterisks (“*”) indicate identical amino acids, while colon (“:”) and period (“.”) indicate conserved and semi-conserved amino acids, respectively. The identical/similar amino acids are shown in shading.
Additional file 4: Figure S3. Alignment of the amino acid sequences of the tryptophan-rich domains (WRDs) of SURFIN 4.1 and SURFIN 4.2 using the MUSCLE program. The remaining variable regions are shown in italic. The putative transmembrane domains (SURFIN 4.1, amino acids 774-793; SURFIN 4.2, amino acids 740-762) are shown in grey colour. The first and second WRDs (SURFIN 4.1: WRD1 positions 908-1141, WRD2 1287-1476; SURFIN 4.2: WRD1 959-1201, WRD2 1349-1567) are shown in yellow colour, and the positionally conserved tryptophan residues are shown in bold and red colour. Asterisks (“*”) indicate identical amino acids while colon (“:”) and period (“.”) indicate conserved and semi-conserved amino acids, respectively. SURFIN 4.1 (accession no. AB759920.1); SURFIN 4.2 (PlasmoDB ID: PF3D7_0424400).
Additional file 5: Figure S4. Preparation of inside-out vesicles (IOVs) from normal human RBCs. ( a) Coomassie Brilliant Blue staining of separated protein present in the IOVs. ( b) Western blot analysis of IOVs. IOVs were prepared and separated by SDS-PAGE, transferred to PVDF membrane, then detected with anti-actin or anti-spectrin antibodies. Recombinant actin was loaded as a positive control. Arrows indicate the spectrin dimer, protein 4.1 and actin.
Additional file 6: Figure S5. Scatchard analyses of the ELISA-based IOV binding assay of WRDs of SURFIN 4.2 and PvSTP2. ( a) Interactions between SURFIN 4.2 WRD1 and IOV. ( b) Interactions between SURFIN 4.2 WRD3 and IOV. ( c) Interactions between PvSTP2 WRD and IOV. ( d) Interactions between Pf332 WRD and IOV.
Additional file 7: Figure S6. Binding of the His-tagged recombinant SURFIN 4.2 WRD2−1 and SURFIN 4.2 CRD to F-actin. ( a) The supernatant and ( b) pellet fractions of SURFIN 4.2 WRD2−1 -His (SURFIN 4.2 WRD2−1 , WRD2-1 1349-1499, calculated MW, 38.0 kDa), SURFIN 4.2 CRD -His (SURFIN 4.2 CRD , CRD 1-197, calculated MW, 42.4 kDa), and His-tag control protein after F-actin co-sedimentation assay were analysed by Western blot with anti-His tag or anti-actin antibodies. Arrows indicate recombinant protein bands with the expected sizes.
Additional file 8: Figure S7. Preparation of the KAHRP spectrin-binding fragment. ( a) Coomassie Brilliant Blue staining of KAHRP spectrin-binding fragment. ( b) Western blot analysis of KAHRP spectrin-binding fragment 320-451. Arrows indicate His-tagged recombinant protein, KAHRP 320−451-His.
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