Proteins Complexed With MSP-

On the merozoite surface, the primary processed 83, 42, 38 and 30 kDa MSP-1

polypeptides are in a non-covalently bound complex with two additional polypeptides o f

22 kDa (MSP-?22) and 36 kDa (MSP-636) (McBride and Heidrich 1987; Stafford et a l

1996a; Trucco et a l 2 0 0 1). These two proteins are not derived from MSP-1, as

determined by N-terminal amino acid sequencing and antibody afiSnity-select experiments

(Stafford et a l 1994).

The shed MSP-1 complex, released after secondary MSP-1 processing, has been

immuno-afSnity purified using an anti-MSP-133 antibody (X509) (see Figure 1.5).

Analysis o f the purified complex suggested that a 19kDa protein (MSP-7iç), the 22 kDa

MSP-722 polypeptide, and a 36kDa polypeptide (MSP-636) are part o f the shed MSP-1

complex (Stafford et a l 1994; Stafford 1996). N-terminal sequencing o f the 19, 2 2 and

36 kDa and MSP-1 polypeptides purified from the shed complex showed that MSP-7i9,

MSP-722, and MSP-636 are distinct from the MSP-1 products o f secondary processing

(Stafford et a l 1994; Stafford et a l 1996a). Two dimensional peptide mapping and

antibody affinity select experiments suggested that MSP-7%9 and MSP-722 were closely

Chapter One : Introduction 47

The gene coding for iyMSP-6 has been identified (Trucco et a l 2001). The msp~6

gene shows no sequence diversity, and codes for a 371-residue MSP-6 precursor (Trucco

et a l 2001). The amino acid sequence o f MSP-6 shows similarity at the N-terminus and

C-terminus to that o f MSP-3 (Trucco et a l 2001). The MSP-6 precursor undergoes N-

terminal cleavage to give the 211-residue, MSP-636, that is associated with the MSP-1

complex. MSP-636 is a highly hydrophilic, negatively-charged protein, that contains a

glutamic acid-rich region and two hydrophobic amino acid clusters that may form part of

its binding site with MSP-1 (Trucco et a l 2001). The cellular location o f the MSP-6

cleavage is unknown, as is the fate o f the N-terminal cleavage product. The lack o f

sequence diversity exhibited by MSP-6 may be due to structural and hence functional

constraints in its interactions with the MSP-1 complex. This suggests that MSP-6 is an

optimal drug and vaccine target, as it may prove to be an essential component in the

MSP-1 complex and hence in erythrocyte invasion.

Cell surface radio-iodination and immunofluorescence experiments suggest that

MSP-?22 is present on the surface o f released merozoites (McBride and Heidrich 1987;

Stafford et a l 1996a). MSP-722 is non-covalently bound to the primary processed MSP-1

complex o f both MAD20 and Wellcome allellic types (McBride and Heidrich 1987;

Stafford et a l 1996a). In the shed MSP-1 complex, MSP-722 is partially cleaved at the N-

terminus into a 19 kDa (MSP-7ig) protein, with MSP-7i9 present in the shed complex at

an estimated 2:1 molar ratio with MSP-722 (Stafford et a l 1996a). The cleavage o f MSP-

722 into MSP-7i9 appears to occur at the same time as secondary processing o f MSP-142,

since MSP-7%9 was detected in the shed MSP-1 complex, but not in primary processed

As MSP-?22 appears to undergo cleavage to MSP-719 at a similar time as MSP-1

undergoes secondary processing, it is possible that the same protease is responsible for

both events. Hence MSP-?22 may be a putative chemotherapy target. As MSP-?22 is

closely associated with MSP-1 on the merozoite surface it may also be the target of

antibodies that inhibit erythrocyte invasion. Hence the biological significance o f MSP-7, in

particular in its interaction with the MSP-1 complex requires determination.

1.8 Aims

In order to study the function of MSP-722, in particular its interaction with MSP-1

and its location on the merozoite, the initial aim for my project was to identify and clone

the msp-7 gene. The partial malaria genome sequence databases were to be searched

using the MSP-722 and MSP-7i9 N-terminal amino acid sequences. To obtain the whole

msp-7 gene, primers were to be designed using retrieved sequences, for use in vectorette

PCR, a form o f one-sided PCR. The msp-7 gene would be cloned and sequenced fi*om

several P. falciparum lines, and analysed by DNA and protein structure programmes.

Regions o f the MSP-7 protein would be expressed as GST or 6*His tagged fusion

proteins in pGEX-3X or pTrcHisC inducible bacterial expression systems. These fusion

proteins would be used to raise specific antibodies against parts o f MSP-7, by

immunisation into mice, and production o f hybridomas expressing monoclonal antibodies.

Antibody specificity would be determined by IF AT on parasitised erythrocytes, and by

immuno-precipitation o f labelled protein.

The time o f msp-7 transcription would be determined by Northern blot analysis.

Chapter One : Introduction 49

analysis, and by immuno-precipitation from lysates o f radiolabelled parasites using

detergents that either disrupt or preserve the MSP-1 complex. The timing and location o f

the association between MSP-1 and MSP-7 would be fiirther studied by dual-immuno-

fluorescence assays, and by immunoprécipitation from lysates o f synchronised parasites

pulse-chased with radiolabelled amino acid precursor. The effect o f Brefeldin A on MSP-

7 transport and its association with MSP-1 would be studied

To determine the fimctional activity o f antibodies to MSP-7, assays would be

performed to determine their effect on MSP-1 secondary processing, and on processing o f

Figure 1.0 The Plasmodium Life Cycle in Humans

A schematic o f the life cycle o f Plasmodium species that infect humans. Taken from a diagram by the Wellcome Trustf The life cycle can be separated into 4 distinct stages: *' (KneU1991)

1. Fertilisation A female anopheline^ mosquito takes a blood meal containing male and female gametocytes from an infected human. (1) The male gametocyte exflagellates, producing up to 8 motile microgametes. (2) These fiise with female macrogametes forming a zygote, which develops into a motile ookinete. (3) The ookinete penetrates the peritrophic membrane and then the midgut wall, and lodges between the outer membrane o f the midgut and the midgut epithelial cells where it develops into an oocyst.

2. Snorogonv (1 - 2) The oocyst undergoes asexual division developing several thousands o f motile sporozoites. (3) After 7 to 20 days the oocyst ruptures and the released sporozoites migrate to the salivary glands where they undergo a brief period of maturation, before entering the salivary ducts.

3. Hepatic Schizogony When the mosquito takes a blood meal sporozoites are injected with saliva into the blood. (1) Sporozoites are transported via the blood stream to the liver where they invade parenchymal cells and develop into pre-erythrocytic schizonts. (2—3) In these the parasite undergoes asexual division into thousands o f invasive merozoites, which after 7 to 20 days are released when the hepatic schizont bursts. (4) The released merozoites enter red blood cells within the hepatic sinusoids to start the asexual intraerythrocytic cycle. (5) Some P. vivax and P. ovale sporozoites develop into hypnozoites, which remain dormant for months or years before undergoing schizogony, resulting in relapsing malaria.

4. Erythrocytic Schizogony (1) Merozoites invade red blood cells, developing into ring stages, and then into trophozoites. (2 - 3) Ring and early trophozoites stages are present in the peripheral circulation. (4 - 5) Trophozoites undergo asexual division (schizogony) developing into schizonts that contain 16 merozoites. Late trophozoites and schizonts of

P. falciparum mature in capillaries o f internal organs. (6 - 7) On schizont rupture the released merozoites reinvade red blood cells continuing the intra-erythrocytic cycle. This cyclical amplification in red blood cells takes 48 hours. (8 - 9) Some merozoites develop into gametocytes, which mature if taken up by a feeding Anopheline mosquito.

2 S P O R O G O N Y