Stafford, William Herbert Lee Stafford; (1996) Analysis of the merozoite surface protein-1 complex and protein secretion in Plasmodium falciparum. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Plasmodium falciparum merozoite surface protein-1 (MSP-1) is synthesized as a precursor of 195kDa and undergoes proteolytic processing. Primary processing produces several fragments held on the surface of the free merozoite as a non-covalently associated complex. Secondary processing results in cleavage of the C-terminal fragment and shedding of the majority of the complex into the extracellular milieu. The MSP-1 complex was affinity purified from culture supernatants and N-terminal amino acid sequencing identified all the MSP-1 (MAD-20 allelic form) primary processing sites. Additionally, three novel polypeptides of 19kDa, 22kDa and 36kDa (p19, p22, and p36 respectively) were associated with the complex. p22 was localized to the merozoite surface by immunofluorescence on unfixed merozoites; confirming its previous localization by merozoite cell surface radioiodination. Several lines of evidence suggest that p19 is derived from p22 by N-terminal proteolysis during or after secondary processing of MSP- 1. The p19/p22 was present in the shed complex in near equimolar amounts to the MSP-1 polypeptides suggesting these interactions are specific. To study p22 in greater detail, considerable efforts were made to clone the relevant gene. This was unsuccessful but led to the serendipitous cloning of a gene with 76% amino acid sequence identity to human ADP-ribosylation factor-1; a small GTP binding protein regulating transport of secretory cargo between the endoplasmic reticulum and Golgi cisternae. The P. falciparum ADP-ribosylation factor gene (Pfarf1) contains four introns and the 543bp exons encode a protein of 20kDa. Pfarf1 localizes to chromosome 10 and the 1.8kb mRNA is developmentally regulated with peak abundance at 36 h. The recombinant protein (PfARF1) was expressed in Escherichia coli. In vitro assays established pfARF1 bound GTP, lacked detectable GTPase activity, and stimulated cholera toxin ADP-ribosyltransferase activity, confirming it as a member of the arf family and functionally distinguishing it from the structurally related arl (arf-like) family.

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