Plasmodium falciparum erythrocyte membrane protein 1

Plasmodium falciparum erythrocyte membrane protein 1 is a family of proteins present on the membrane surface of red blood cells that are infected by the malarial parasite Plasmodium falciparum. PfEMP1 is synthesized during the parasite's blood stage inside the RBC, during which the clinical symptoms of falciparum malaria are manifested. Acting as both an antigen and adhesion protein, it is thought to play a key role in the high level of virulence associated with P. falciparum. It was discovered in 1984 when it was reported that infected RBCs had unusually large-sized cell membrane proteins, and these proteins had antibody-binding properties. An elusive protein, its chemical structure and molecular properties were revealed only after a decade, in 1995. It is now established that there is not one but a large family of PfEMP1 proteins, genetically regulated by a group of about 60 genes called var. Each P. falciparum is able to switch on and off specific var genes to produce a functionally different protein, thereby evading the host's immune system. RBCs carrying PfEMP1 on their surface stick to endothelial cells, which facilitates further binding with uninfected RBCs, ultimately helping the parasite to both spread to other RBCs as well as bringing about the fatal symptoms of P. falciparum malaria.

Introduction

Malaria is the deadliest among infectious diseases, accounting for approximately 429,000 human deaths in 2015 as of the latest estimate by the World Health Organization. In humans, malaria can be caused by five Plasmodium parasites, namely P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. P. falciparum is the most dangerous species, attributed to >99% of malaria's death toll, with 70% of these deaths occurring in children under the age of five years. The parasites are transmitted through the bites of female mosquitos. Before invading the RBCs and causing the symptoms of malaria, the parasites first multiply in the liver. The daughter parasites called merozoites then only infect the RBCs. They undergo structural development inside the RBCs, becoming trophozoites and schizonts. It is during this period that malarial symptoms are produced.
Unlike RBCs infected by other Plasmodium species, P. falciparum-infected RBCs had been known to spontaneously stick together. By the early 1980s, it was established that when the parasite enters the blood stream and infects RBCs, the infected cells form knobs on their surface. Then they become sticky, and get attached to the walls of the blood vessels through a process called cytoadhesion, or cytoadherence. Such attachment favours binding with and accumulation of other RBCs. This process is known as sequestration. It is during this condition that the parasites induce an immune response and evade destruction in the spleen. Although the process and significance of sequestration were described in detail by two Italian physicians Amico Bignami and Ettore Marchiafava in the early 1890s, it took a century to discover the actual factor for the stickiness and virulence.

Discovery

PfEMP1 was discovered by Russell J. Howard and his colleagues at the US National Institutes of Health in 1984. Using the techniques of radioiodination and immunoprecipitation, they found a unique but yet unknown antigen from P. falciparum-infected RBCs that appeared to cause binding with other cells. Since the antigenic protein could only be detected in infected cells, they asserted that the protein was produced by the malarial parasite, and not by RBCs. The antigen was large and appeared to be different in size in different strains of P. falciparum obtained from night monkey. In one strain, called Camp, the antigen was found to have a molecular size of approximately 285 kDa; while in the other, called St. Lucia, it was approximately 260 kDa. Both antigens bind to cultured skin cancer cells. But the researchers failed to confirm whether or not the protein actually was an adhesion molecule to the wall of blood vessels. Later in the same year, they found out that the unknown antigen was associated only with RBCs having small lumps called knobs on their surface. The first human RBC antigen was reported in 1986. Howard's team found that the antigens from Gambian children, who were suffering from falciparum malaria, were similar to those from the RBCs of night monkey. They determined that the molecular sizes of the proteins ranged from 250 to 300 kDa.
In 1987, they discovered another type of surface antigen from the same Camp and St. Lucia strains of malarial parasites. This was also a large-sized protein of about 300 kDa, but quite different from the antigens reported in 1984. The new protein was unable to bind to melanoma cells and present only inside the cell. Hence, they named the earlier protein Plasmodium falciparum erythrocyte membrane protein 1, to distinguish it from the newly identified Plasmodium falciparum erythrocyte membrane protein 2. The distinction was confirmed the next year, with an additional information that PfEMP1 is relatively less in number.
Although some of the properties of PfEMP1 were firmly established, the protein was difficult to isolate due to its low occurrence. Five years after its discovery, one of the original researchers Irwin Sherman began to doubt the existence of PfEMP1 as a unique protein. He argued that the antigen could be merely a surface protein of RBCs that changes upon infection with malarial parasites. A consensus was achieved in 1995 following the identification of the gene for PfEMP1. The discovery of the genes was independently reported by Howard's team and two other teams at NIH. Howard's team identified two genes for PfEMP1, and recombinant protein products of these genes were shown to have antigenic and adhesive properties. They further affirmed that PfEMP1 is the key molecule in the ability of P. falciparum to evade the host's immune system. Joseph D. Smith and others showed that PfEMP1 is actually a large family of proteins encoded by a multigene family called var. The gene products can bind to a variety of receptors including those on endothelial cells. Xin-Zhuan Su and others showed that there could be more than 50 var genes which are distributed on different chromosomes of the malarial parasite.

Structure

PfEMP1 is a large family of proteins having high molecular weights ranging from 200 to 350 kDa. The wide range of molecular size reflects extreme variation in the amino acid composition of the proteins. But all the PfEMP1 proteins can be described as having three basic structural components, namely, an extracellular domain, a transmembrane domain and an intracellular acidic terminal segment. The extracellular domain is fully exposed on the cell surface, and is the most variable region. It consists of a number of sub-domains, including a short and conserved N terminal segment at the outermost region, followed by a highly variable Duffy-binding-like domain, sometimes a Ca²⁺-binding C2 domain, and then one or two cysteine-rich interdomain regions.
Duffy-binding-like domains are so named because of their similarity to the Duffy binding proteins of P. vivax and P. knowlesi. There are six variant types of DBL, named DBLα, DBLβ, DBLγ, DBLδ, DBLε and DBLζ. CIDR is also divided into three classes: CIDRα, CIDRβ and CIDRγ. Both DBL and CIDR have an additional type called PAM, so named because of their specific involvement in pregnancy-associated malaria. In spite of the diverse DBL and CIDR proteins, the extracellular amino terminal region is partly conserved, consisting of about 60 amino acids of NTS, one each of DBLα and CIDR1 proteins in tandem. This semi-conserved DBLα-CIDR1 region is called the head structure. The last CIDR region joins the TMD, which is embedded in the cell membrane. The TMD and ATS are highly conserved among different PfEMP1s, and their structures have been solved using solution NMR.
The head structure is followed by a variable combination of diverse DBL and CIDR proteins, and in many cases along with C2. This variation gives rise to different types of PfEMP1. The DBL-CIDR combination in a particular type of PfEMP1 protein is never random, but organized into specific sequences known as domain cassettes. In some domain cassettes, there are only two or few DBL domains and CIDR domains, but in others they cover the entire length of the PfEMP1. These differences are responsible for different binding capacity among different PfEMP1s. For instance, among the most well-known types, VAR3 is the smallest, consisting of only NTS with DBL1α and DBL2ε domains in the ECD. Its molecular size is approximately 150 kDa. In domain cassette 4 type, the ECD is made up of three domains DBLα_1.1/1.4, CIDRα_1.6 and DBLβ₃. The DBLβ₃ domain contains a binding site for intercellular adhesion molecule 1. This is particularly implicated with the development of brain infection. VAR2CSA is atypical in having a single domain cassette that consists of three N terminal DBL_PAM domains followed by three DBLε domains and one CIDR_PAM. The seven domains always occur together. The usual NTS is absent. The protein specifically binds to chondroitin sulphate A ; hence the name VAR2CSA.

Synthesis and transport

The PfEMP1 proteins are regulated and produced by about 60 different var genes, but an individual P. falciparum would switch on only a single var gene at a time to produce only one type of PfEMP. The var genes are distributed in two exons. Exon 1 encodes amino acids of the highly variable ECD, while exon 2 encodes those of the conserved TMD and ATS. Based on their location in the chromosome and sequence, the var genes are generally classified into three major groups, A, B, and C, and two intermediate groups, B/A and B/C; or sometimes simply into five classes, upsA, upsB, upsC, upsD, and upsE respectively. Groups A and B are found towards the terminal end region of the chromosome, while group C is in the central region.
Once the PfEMP1 protein is fully synthesized, it is carried to the cytoplasm towards the RBC membrane. The NTS is crucial for such directional movement. Within the cytoplasm, the newly synthesized protein is attached to a Golgi-like membranous vesicle called the Maurer's cleft. Inside the Maurer's clefts is a family of proteins called Plasmodium helical interspersed subtelomeric proteins. Of the PHIST proteins, PFI1780w and PFE1605w bind the intracellular ATS of PfEMP1 during transport to the RBC membrane.
The PfEMP1 molecule is deposited at the RBC membrane at the knobs. These knobs are easily identified as conspicuous bumps on the infected RBCs from the early trophozoite stage onward. The malarial parasite cannot induce its virulence on RBCs without knobs. As many as 10,000 knobs are distributed throughout the surface of a mature infected RBC, and each knob is 50-80 nm in diameter. The export of pfEMP1 from Maurer's cleft to RBC membrane is mediated by binding of another protein produced by the parasite called knob-associated histidine-rich protein. KAHRP enhances the structural rigidity of infected RBC and adhesion of PfEMP1 on the knobs. It is also directly responsible for forming knobs, as indicated by the fact that kahrp gene-deficient malarial parasites do not form knobs. To form a knob, KAHRP aggregates several membrane skeletal proteins of the host RBC, such as spectrin, actin, ankyrin R, and spectrin–actin band 4.1 complex. Upon arrival at the knob, PfEMP1 is attached to the spectrin network using the PHIST proteins.