Identification and Stoichiometry of Glycosylphosphatidylinositol-anchored Membrane Proteins of the Human Malaria Parasite Plasmodium falciparum

Paul R Gilson,Thomas Nebl,Damjan Vukcevic,Robert L Moritz,Tobias Sargeant,Terence P Speed,Louis Schofield,Brendan S Crabb

doi:10.1074/mcp.m600035-mcp200

Abstract

Most proteins that coat the surface of the extracellular forms of the human malaria parasite Plasmodium falciparum are attached to the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. These proteins are exposed to neutralizing antibodies, and several are advanced vaccine candidates. To identify the GPI-anchored proteome of P. falciparum we used a combination of proteomic and computational approaches. Focusing on the clinically relevant blood stage of the life cycle, proteomic analysis of proteins labeled with radioactive glucosamine identified GPI anchoring on 11 proteins (merozoite surface protein (MSP)-1, -2, -4, -5, -10, rhoptry-associated membrane antigen, apical sushi protein, Pf92, Pf38, Pf12, and Pf34). These proteins represent approximately 94% of the GPI-anchored schizont/merozoite proteome and constitute by far the largest validated set of GPI-anchored proteins in this organism. Moreover MSP-1 and MSP-2 were present in similar copy number, and we estimated that together these proteins comprise approximately two-thirds of the total membrane-associated surface coat. This is the first time the stoichiometry of MSPs has been examined. We observed that available software performed poorly in predicting GPI anchoring on P. falciparum proteins where such modification had been validated by proteomics. Therefore, we developed a hidden Markov model (GPI-HMM) trained on P. falciparum sequences and used this to rank all proteins encoded in the completed P. falciparum genome according to their likelihood of being GPI-anchored. GPI-HMM predicted GPI modification on all validated proteins, on several known membrane proteins, and on a number of novel, presumably surface, proteins expressed in the blood, insect, and/or pre-erythrocytic stages of the life cycle. Together this work identified 11 and predicted a further 19 GPI-anchored proteins in P. falciparum.

Highlights

Most proteins that coat the surface of the extracellular forms of the human malaria parasite Plasmodium falciparum are attached to the plasma membrane via glycosylphosphatidylinositol (GPI) anchors
The preprotein sequences of GPI-anchored proteins are characterized by being book-ended by an N-terminal hydrophobic signal sequence that directs co-translational insertion into the endoplasmic reticulum (ER) and a C-terminal hydrophobic region that guides the attachment of a GPI anchor
To predict the identity of these lower abundance proteins as well as those present at other life stages we developed a program, GPI-HMM, trained on P. falciparum sequences to search all annotated and predicted protein sequences derived from the P. falciparum genome

Summary

Introduction

Most proteins that coat the surface of the extracellular forms of the human malaria parasite Plasmodium falciparum are attached to the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. The relative amount of GPI-anchored protein was calculated from densitometric scans of autoradiographs of [3H]glucosamine-labeled total schizont proteins separated by high resolution 1D gel electrophoresis.

Objectives

Results

Conclusion