3.5 9.089E-03 PF3D7_1010200 PF10_0099 DNA2 helicase,

putative -1.33 0.13 PF3D7_0202000 Knob-associated histidine-rich protein (KAHRP) Knob formation; deformability 4.06 0.14 PF3D7_1004000 PF10_0043 60S ribosomal protein L13, putative 1.13 0.42

PF3D7_0917900 Heat shock protein (Hsp70-2) Chaperone 0.76 0.43 PF3D7_0824500 PF08_0029 Unknown 0.48 0.54 PF3D7_0704600 E3 ubiquitin-protein ligase Ubiquitination 0.75 0.74 PF3D7_0532400 Lysine-rich membrane- associated PHISTb protein (LyMP) Cytoadherence 0.75 0.79

PF3D7_1357000 Elongation factor 1- Protein

biosynthesis 0.21 0.84 PF3D7_1310400 PF13_0056 Unknown 0.16 0.85 PF3D7_1462800 Glyceraldehyde-3- phosphate dehydrogenase Metabolic 0.07 0.96

Table 3-1 The MaxQuant analysis of the PFB0115w-GFP complex to determine parasite protein

co-precipitates that showed a fold increase in the PFB0115w-GFP precipitated complex. Proteins that showed a significant fold change (p<0,05) are highlighted in purple. The bait protein PFB0115w-GFP is highlighted in red.

Protein name Function Log Fold-

change Adjusted p-value Human origin

Catalase Oxidative stress 7.67 9.92E-05

Ankyrin-1 7.14 1.31E-05

Spectrin beta chain, erythrocytic B Cytoskeleton chain component 4.83 8.37E-05 Spectrin alpha chain, erythrocytic 1 Cytoskeleton chain component 5.39 0.06 Tubulin alpha-1B/C chain

Microtubule

protein 2.06 0.07

Polyubiquitin-B Protein degradation 1.41 0.08

Band 3 anion transport protein Cytoskeleton junction protein 2.575 0.15 Glucose-regulated protein 78 Human chaperone protein 0.97 0.26

Calpain-1 Cysteine protease 1.52 0.41

Protein 4.1 Cytoskeleton junction protein 1.49 0.41 Zymogen granule protein 16 homolog Carbohydrate binding, putative -1.56 0.42

POF1B Actin-binding protein 0.98 0.42

Xin actin-binding repeat-containing protein 1 Actin-binding protein -0.96 0.42

Histone H2A.V Chromatin structure -0.63 0.51

Annexin A2

Pleiotropic,

membrane bound 0.61 0.51

Table 3-2 The MaxQuant analysis of the PFB0115w-GFP complex to determine human host

protein co-precipitates that showed a fold increase in the PFB0115w-GFP precipitated complex. The top 15 proteins as ranked by p-value are shown here. Proteins that showed a significant fold change (p<0,05) are highlighted in green.

Chapter 3

Several proteins of both parasite and human origin showed a significant fold change(p< 0.05) as summarised in Figure 3.5.The parasite proteins that showed the greatest fold change (p < 0.005) were PFD80 (PF3D7_0401800) and the mature erythrocyte surface antigen, MESA (PF3D7_0500800), which showed a 230-fold and 170-fold increase respectively, as compared to the wildtype control. Glycophorin binding protein, GBP1 (PF3D7_1016300) also showed a significant fold increase (p < 0.01) with a 15-fold increase in the PFB0115w-GFP precipitate as compared to the wildtype control.

Figure 3-5 Volcano plot of proteins that showed a fold increase in PFB0115w precipitated complex

over untagged background control (CS2 WT). Proteins that show a significant fold change (p < 0.05) are highlighted in either purple for parasite proteins or green for human host proteins. The bait protein PFB0115w-GFP is shown in red.

Although MaxQaunt can identify co-precipitates with a significant fold change, the software is limited in its scope to identify proteins that have a relatively low abundance in the PFB0115w-GFP immunoprecipitated complex but are unique to the complex. Thus, we also used Scaffold, an analytical software that categorises a protein as either present or absent and

Protein ID Common Name Parasite proteins

PF3D7_0202400 PFB0115w

PF3D7_0730900 PfEMP1-trafficking protein 1 (PTP4) PF3D7_ PF3D7_0201900 Erythrocyte membrane protein 3 (PfEMP3)

PF3D7_0818900 Heat shock protein 70

PF3D7_0532300 PFE1600w, uncharacterised PHISTb protein

PF3D7_0401800 PFD80

PF3D7_0500800 Mature erythrocyte surface antigen (MESA) PF3D7_1016300 Glycophorin binding protein (GBP) PF3D7_0202000 Knob-associated histidine-rich protein (KAHRP)

Human proteins

ANK3 Ankyrin-3

SPTB1 Spectrin -chain 1, erythrocytic

SPTA1 Spectrin -chain 1, erythrocytic

TUBA1C Tubulin 1c chain

PRDX2 Peroxiredioxin-2

EEF2 Elongation factor 2

HUTH Histidine ammonia lyase

CSTA Cystatin A

CDSN Corneodesmosin

ECM1 Extracellular matrix protein 1

Table 3-3 Scaffold analysis of the PFB0115w-GFP precipitated complex compared to the wildtype CS2 control. Bait protein (PFB0115w) highlighted in red. Proteins unique to the Scaffold dataset as compared to the MaxQuant dataset highlighted in orange. Proteins common between Scaffold and MaxQuant dataset shown in white.

There are several proteins that are observed in both Maxquant and Scaffold datasets. This overlap is due to the differences in the analytical methods used by the two pieces of software which results in a distinction in the background level estimation. Three of the four overlapping parasite proteins also showed a significant (p<0.05) increase in abundance (PFD80, MESA and GBP), which indicates that abundance of these proteins is likely to be negligible in the wildtype control and thus, they are unique highly abundant co-precipitates of PFB0115w-GFP. Additionally, a majority of human proteins observed in the Scaffold dataset were also found in the MaxQuant dataset however all proteins, expect for Spectrin -chain 1, did not show a significant (p<0.05) fold change.

There were an additional five proteins that were unique to the Scaffold dataset: PFE1600w (PF3D7_0532300, an unknown PHISTb protein; PTP4, a Maurer’s cleft protein; PfEMP3 (PF3D7_0201900), a virulence complex protein, PfHsp70-1 (PF3D7_0818900), a DnaJ chaperone protein implied in the export of virulence factors and ankyrin 3, a human

Chapter 3

Figure 3-6 Analysis of detergent solubilised

PfHsp70-1 and MESA with Native PAGE A) GFP- tagged PfHsp70-1, solubilised in 2% Zwittergent, did not show any presence of a specific complex as compared to the untagged wildtype (CS2 WT) control. The blot was probed with anti-GFP antibody. B) HA-tagged MESA, solubilised in 2% Zwittergent, was detected in a 500 kDa band as compared to the untagged MESA (CS2 WT) control. The blot was probed with anti-HA antibody.

The potential protein interactions with PFB0115w were then further investigated for MESA and PfHsp70-1 (Figure 3.6) using a reciprocal approach: parasites with either HA-tagged MESA (CS2_MESA-HA) and GFP-tagged PfHsp70-1 were purified and ghost lysates from these cells were treated with 2% (w/v) Zwittergent in the same manner as that for PFB0115w. Solubilised lysates were then assayed on a native PAGE gel to visualise the presence of complexes, if any. In the case of PfHsp70-1, all observed bands were also present in the wild type control indicating a non-specific cross reaction from the GFP antibody (Figure 3.6 A). Solubilised MESA however appeared in a band at just above 480 kDa, a similar size to that of the PFB0115 solubilised complex (Figure 3.6 B). This is strong evidence to suggest MESA does indeed interact with PFB0115w however a reciprocal co-immunoprecipitation with MESA is required to confirm the interaction.

Figure 3-7 Western blot analysis of the immunoprecipitated PFB0115w-GFP complex compared

to the wildtype untagged control. Blot was initially probed with anti-GFP antibody against bait protein PFB0115w and then sequentially with antibodies specific to target proteins ankyrin-1 and spectrin 1. The red arrow indicates the expected full-length size of protein probed by the specific antibody. CL stands for ghost lysate solubilised with 2% (w/v) Zwittergent 3-14; UB stands for unbound protein flowthrough and B denotes bound protein fraction post chloroform methanol extraction from beads.

The mass spectrometric analysis also revealed three host proteins to be significantly more abundant (p < 0.0001) in the PFB0115w-GFP complex: Catalase, Ankyrin-1 and Spectrin 1. Catalase is an oxidative stress activated protein that is abundant in erythrocytes and its association with PFB0115w-GFP was not pursued further here due to its ubiquitous presence. The presence of ankyrin-1 and spectrin 1, both components of the host cell cytoskeleton, could be indicative of a host-parasite interaction. In order to investigate this further, western blot analysis (Figure 3.7) of the eluted bound fraction against the unbound and total lysate fractions was conducted. It revealed that both Ankyrin and spectrin 1 do not associate with the complex, since they were not present in the bound fraction. The complex was also probed for PFB0115w-GFP to confirm the successful immunoprecipitation of the complex but showed decreased abundance of PFB0115w-GFP as compared to the total cell lysate (CL). This might have been due to incomplete elution from the beads by the chloroform methanol extraction, as western blot analysis of the beads post-elution showed that PFB0115w was not completely eluted (not shown here).

Chapter 3

PFB0115w does not present on the host cell membrane

Surface presentation of PFB0115w on the host cell membrane was tested through a trypsin cleavage assay and compared to the known surface antigen PfEMP1 (Leech et al. 1984). Membranes of trypsin-treated infected erythrocytes were obtained either through ghost extraction or through Triton X-100 and SDS solubilisation.

PFB0115w-GFP was unaffected by the presence or absence of trypsin in both ghost and TX- 100 insoluble/SDS soluble lysate and appeared only in its full-length form. This indicates that trypsin was unable to cleave PFB0115w-GFP (Figure 3.8) and hence PFB0115w-GFP is not exposed on the host cell membrane. The host cell surface exposed PfEMP1 was used as a positive control and was cleaved in the presence of trypsin to produce a band below 100 kDa, which is consistent with previous trypsin assays(Rug et al. 2014).

Figure 3-8 Trypsin cleavage of PFB0115w-

GFP in comparison to the surface antigen PfEMP1. The ghost fraction and Triton X100 insoluble/SDS soluble (TX-100/SDS) fraction of whole cell lysates of CS2PFB0115w-GFP were either probed with anti-GFP antibody or anti-ATS antibody, which is specific to the cytoplasmic segment of PfEMP1. Cells were treated with either PBS (-) as the negative control or with PBS and trypsin (+). The green arrow indicates the expected full-length size of PFB0115-GFP, the black arrow indicates the full-length size of PfEMP1, the purple arrow indicates the size of spectrin, a product of cross reactivity of the anti-ATS antibody, the red arrow indicates the size of the trypsin cleaved segment of PfEMP1. This experiment was representative of three independent experiments

.