Decoding the language of var genes and Plasmodium falciparum sequestration

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Decoding the language of var genes and Plasmodium falciparum sequestration Joseph D. Smith, Benoit Gamain, Dror I. Baruch and Sue Kyes Sequestration and rosetting are key determinants of Plasmodium falciparum pathogenesis. They are mediated by a large family of variant proteins called P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are multispecific binding receptors that are transported to parasite-induced, ‘knob-like’ binding structures at the erythrocyte surface. To evade immunity and extend infections, parasites clonally vary their expressed PfEMP1. Thus, PfEMP1 are functionally selected for binding while immune selection acts to diversify the family. Here, we describe a new way to analyse PfEMP1 sequence that provides insight into domain function and protein architecture with potential implications for malaria disease.

Joseph D. Smith* Dept of Pathology, Colorado State University, Fort Collins, CO 80523, USA. *e-mail: joseph.smith@ colostate.edu Benoit Gamain Dror I. Baruch Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Sue Kyes Molecular Parasitology Group, Institute of Molecular Medicine, Nuffield Dept of Medicine, John Radcliffe Hospital, Headington, Oxford, UK OX3 9DU.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a large family of variant proteins that have a central role in P. falciparum biology and the host–parasite interaction1,2. Infected erythrocyte cytoadherence is a critical process for parasite survival and transmission, and contributes significantly to P. falciparum virulence and disease pathogenesis. Antigenic variation is an important strategy for the parasite to evade host immunity and establish chronic infections. As adhesion and antigenic variation properties reside in the large and diverse var gene family that encode PfEMP1, it is important to understand how they are simultaneously maintained and evolve. A challenge to understanding the parasite’s functional capabilities is the extreme sequence diversity and complicated gene regulation of the PfEMP1 family. This complexity is being partially deciphered through sequence analysis. Structure of the var genes and PfEMP1 proteins

The var genes comprise two exons3. Exon 1 codes for the variable extracellular domain and transmembrane region, and exon 2 for a highly conserved cytoplasmic or acidic terminal segment (ATS). Most var genes are located in subtelomeric regions on both ends of the parasite’s 14 chromosomes4–6. In addition, chromosomes 4, 7, 12 and perhaps 8 contain var genes that are centrally located6. When they were first described3,7,8, two domains figured prominently in the PfEMP1 extracellular region: the DUFFY-BINDING-LIKE DOMAIN (DBL; see Glossary) and the CYSTEINE-RICH INTERDOMAIN REGION (CIDR) (Fig. 1). Whereas the CIDR domain was a novel structure that is unique to P. falciparum, the DBL domain was a familiar element that had been shown to function as an adhesive module in the http://parasites.trends.com

erythrocyte-binding antigens (EBAs) of several malaria species, which are used during parasite invasion of erythrocytes9–11. Because erythrocyte invasion is a fundamental property required for survival and transmission of all Plasmodium species and SEQUESTRATION is restricted to only some, PfEMP1 proteins are thought to have evolved from the EBAs. The creation of PfEMP1 and development of a capacity for infected erythrocyte cytoadherence are believed to be major factors contributing to the special virulence of P. falciparum. The original PfEMP1 proteins described were similar, in that each began with a ‘semiconserved head structure’ consisting of a DBL and CIDR domain, which was followed by one or more DBL domains3 (Fig. 1). Furthermore, even though the DBL domains were highly diverse, they grouped into four different sequence classes with the arrangement of internal DBL types differing between different PfEMP1 proteins. Recently, we reanalyzed PfEMP1 sequences in order to characterize the undescribed regions and to place DBL domain classifications on a firmer molecular foundation12 (S. Kyes, unpublished). This analysis offers a new way to describe and classify PfEMP1 domains, and introduces a universal language to discuss and compare findings. Coupled with progress in defining PfEMP1 adhesive domains, a clearer molecular explanation has begun to emerge of the many binding properties encoded within the PfEMP1 protein family. The PfEMP1 extracellular framework is predominantly composed of four basic building blocks: an N-TERMINAL SEGMENT (NTS), DBL domains, CIDR domains and C2 DOMAINS (Fig. 1). Each domain has characteristic attributes3,12,13. The NTS is predicted to be globular with a relatively conserved central α-helical fold. The NTS contains between 75 and 107 residues including a conserved cysteine residue. Interestingly, the NTS lacks a conventional signal sequence, confusing the issue of how PfEMP1 proteins are exported to the erythrocyte surface. It is also not known if the NTS region is present in the mature protein. C2 domains vary in length between 140 and 217 residues, are predicted to be globular with regions of α-helical structure and have four invariant cysteine residues.

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Glossary Duffy-binding-like domain (DBL): Plasmodium adhesive domain present both in PfEMP1 and erythrocyte binding antigens used during invasion. First identified in the Plasmodium knowlesi Duffy-antigen-binding protein9. Cysteine-rich interdomain region (CIDR): Adhesive domain present in one to two copies per PfEMP1 protein, characterized by 13 conserved cysteine residues including those belonging to a ‘cysteine-rich motif’ (Fig. 2b,c).

Sequestration: Process in which erythrocytes infected by mature asexual and immature sexual stage parasites bind microvascular endothelium, placenta or bone marrow and remove themselves from blood circulation. N-terminal segment: Semiconserved region at the beginning of all PfEMP1 proteins. C2 domain: PfEMP1 domain that has been universally found tandemly arranged with DBLβ type domains (Fig. 1).

DBL domains, in both PfEMP1 and EBA proteins, have as a unifying feature ten invariant cysteine residues (Fig. 2a). The cysteines are unevenly distributed among ten semiconserved homology blocks (A–J) in which DBL conservation is significantly concentrated (Fig. 3). That is, the homology blocks include long stretches (up to 20 amino acids) in which residues are invariant or conserved for character. Homology blocks of different DBL sequences are predicted to have similar secondary structure, supporting the concept of a DBL fold. Wrapped in the DBL fold is a vast diversity of sequences, with a large proportion of differences occurring in the hypervariable blocks (I–X) that flank the homology blocks (Fig. 3b). Thus, DBL domains maintain dispersed stretches of relatively clustered amino acid conservation. CIDR domains, like DBL domains, are predicted to have a related fold that tolerates extensive sequence variation. By comparison, they have less conspicuous homology blocks but still maintain semiconserved regions with residues that are invariant or have a

Rosetting: Binding of infected erythrocytes to uninfected erythrocytes. This is a property of only a subset of parasite clones. ‘Knob-like’ membrane protrusion: Electron-dense protrusion underneath the infected erythrocyte membrane. A complex of parasite proteins and erythrocyte cytoskeleton elements that acts as the point of attachment for infected-erythrocyte cytoadherence.

fixed amino acid character, such as the cysteine-rich motif (Fig. 2b,c). Conserved CIDR hallmarks include 13 invariant cysteine residues. These features have been used to show that a PfEMP1 domain referred to in some publications as the segment of variable length (SVL) is actually a second CIDR domain present in many PfEMP1 proteins (Figs 1,2b,c). Although PfEMP1 proteins are highly diverse, an important concept, supported by sequence analysis, is that DBL and CIDR domains group into a limited number of distinct sequence classes whose members have greater relatedness. DBL domains form five types (α, β, γ, δ and ε) and CIDR domains three types (α, β and γ) that are statistically distinct by bootstrapping methods12. The different sequence classes have unique type-specific consensus motifs that can be used to characterize future cloned PfEMP1 proteins12. As the number, location and type of DBL and CIDR domains vary among PfEMP1 proteins, we introduced a nomenclature that describes both the numeric position and the sequence type of the domains (Fig. 1).

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Fig. 1. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) protein architecture and binding domains. (a,b) PfEMP1 family members are variably sized proteins that make a single pass through the erythrocyte membrane. The intracellular domain ATS is highly conserved and anchors PfEMP1 to parasite-induced, ‘KNOB-LIKE’ MEMBRANE PROTRUSIONS that facilitate infected erythrocyte sequestration7,56–58. The extracellular domain is highly variable but is predominantly assembled from four building blocks: NTS, DBL, CIDR and C2 domains. Based on sequence similarity, DBL domains group as five types (α–ε) and CIDR domains as three types (α–γ)12. DBLα, β and δ types pair with other domains. The prototypical PfEMP1 extracellular region (a) consists of an NTS and DBL1α–CIDR1 ‘semiconserved head

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structure’ followed by a DBL2δ–CIDR2 tandem. Larger PfEMP1 proteins (b) also include the DBLβ, γ and ε types arrayed differently. Mapped binding traits19–27,33 are indicated with the domain and sequence class that is bound. CD36 is considered to be the major endothelial sequestration receptor; other receptors are recognized less frequently but might have important roles in disease. The CIDR binding region for chondroitin sulfate A/chondroitin sulfate C (CSA/C) is italicized because the parasite only bound CSA (Ref. 24). Abbreviation: CR1, complement receptor 1. Abbreviations: ATS, acidic terminal segment; CIDR, cysteine-rich interdomain region; DBL, Duffy-binding-like domain; ICAM-1, intracellular adhesion molecule-1; NTS, N-terminal segment; TM, transmembrane domain.

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Fig. 2. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) conservation and polymorphism. (a) Duffy-binding-like domain (DBL) sequence conservation is significantly concentrated in ten semiconserved homology blocks (colored pink and shown as the same size for simplicity) flanked by ten hypervariable blocks (blue). DBL domains display type-specific differences in length and molecular mass. Size variation is illustrated as the average length difference between DBLα and δ types. Distributed unequally among homology blocks are ten conserved cysteine residues (C1–C10) present both in PfEMP1 and erythrocyte-binding antigen (EBA) DBL domains. Additional type-specific cysteine residues are also present12. Crucial EBA DBL binding residues map between C4 and C7 (Ref. 53). (b) Cysteine-rich intracellular domain region (CIDR) domains can be divided into three areas (M1, M2 and M3) based on the Malayan Camp CIDR1, in which the minimal CD36 binding region (M2) has been defined. M1 and M2 areas with greater sequence conservation are indicated with bold black lines. The M3 area is extremely degenerate (indicated with green background) and frequently contains runs of charged residues. The general position of 13 conserved cysteine residues (C1–C13) are shown for CIDRα and β types. Additional typespecific cysteines are colored orange (CIDRα) or green (CIDRβ). (c) Comparisons of CIDRα and β consensus motifs, with invariant cysteine residues numbered.

PfEMP1 protein architecture

The sequence approach described above offers insights into PfEMP1 domain assembly and protein architecture. PfEMP1 proteins are not randomly assembled from their building blocks. Rather, there is a strong tendency for domains to occupy certain positions and to associate in favored pairings. Thus, there appear to be higher-order principles governing PfEMP1 structure. Three DBL types, α, β and δ, assemble with other domains (Fig. 1). In addition, the region following most DBLγ and ε types is similar, with four conserved cysteines, but is otherwise more degenerate (J.D. Smith, unpublished). One tandem, DBL1α–CIDR1α, is a general feature of the semiconserved head structure initially described by Su et al.3 The other tandems, DBLβ–C2 and DBLδ–CIDR2(β/γ) are found internal to the head structure. Strikingly, tandem associations are extremely conserved, despite the high rate of PfEMP1 http://parasites.trends.com

recombination14,15. The only exception is that a few DBLβ domains are not coupled to C2 domains. Although individual CIDR or DBL domains expressed as recombinant proteins have frequently been shown to be biologically active, these associations suggest that PfEMP1 domains are not simply modular units that can be interchangeably shuffled, but might also function as more complex tandems with linked structural or functional properties. Using the sequence approach, important distinctions can be observed between PfEMP1 proteins in their size and domain composition. The prototypical PfEMP1 extracellular region consists of an NTS domain followed by a duplicated arrangement of the DBL–CIDR tandem (Fig. 1a). The first tandem, or head structure, is nearly always DBL1α–CIDR1α, and the second is generally DBLδ–CIDRβ. This basic module describes virtually all small PfEMP1 proteins. Large PfEMP1 proteins build on this prototype by adding DBLβ, γ and ε types between or after the duplicated tandem, with occasional PfEMP1 proteins lacking the second tandem12 (Fig. 1b). DBLβ, γ and ε types can have special binding properties and functionally distinguish large PfEMP1 proteins from the prototype. PfEMP1 binding domains

The PfEMP1 structure, with its variable domain composition and extensive sequence polymorphism provides great flexibility in binding. It is now clear that host receptors synergize for parasite adhesion16,17, with some parasite clones binding many receptors18. A current priority is to understand which receptors are most important to parasite survival and human disease. Data have been presented suggesting that at least eight different receptors can be recognized by the PfEMP1 head structure19–26 (Fig. 1). Head-structure binding traits participate in both sequestration and

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hRRLHlCcpNLEphps....stp...LL...-VhhAApaEGpSl.t.a...ptp......uphCThLARSFADI consensus/80% SD102A/5817420 DKNIQQIKAE-QIT-T-HNLL--ADVCMAAKFEGQSISGYHPQYDATYPGS-GSTMCTML K156b/2252527 DRNLEQIDPA-KIT-T-HNLL--LDVCLAAKFEGQSISGYHPQYDD----S-GSTMCTML SD106E/5817486 DQHLSHMKAEKINSKD--NLL--LEVCLAAQYEGESLVEKHKEFKK---THNDSNIYTIL K162d/2252531 DQHLSHMQADKINTKD--NLL--LEVCLAAQYEGQSIRVDHDKYKLD-KDNSGSKLCTEL Van4a/2252534 DQHLSHMQADKINTKD--NLL--LEVLLAAKYEGQSIRVDHDKYKQ---SNNDSQICTAL SD105k/5817456 DHHLSYMNAGKTNTTD--NLL--LEVCMAAQYEGQSIRGQHDKHKLD-NNNSSSQLCTVL SD126A/5817492 DQHLSHMKAEKIKN--KHNLL--LEVCLAAKHEGQSIAGQHGKYHTDGSG---STMCTVL SD126G/5817504 VRNLENISALHKINNHT--LLA--DVCLAALHEGAAISRNHGKHQLTN---SYSQLCTEL SD126J/5817508 VRNLENISALHKINNHT--LLA--DVCLAALHGGAAISRNHGKHQLTN---SYSQLCTEL SD102C/5817424 VRNLENISALDKINNDT--LLA--DVCLAALHEGAAIRGDHGQYQETN---EGSQLCTML SD105N/5817464 VRNLENISNYGKINNDT--LLA--DVCLAALHEGAAISSDHGKYQETNND-VNANICTML SD106G/5817490 VRNLENISALDKINNDT--LLA--DVCLAALHEGQSITQDYPKYQAQYASSSPSQICTML Vnm13E/2252543 VRNLENISNYEKINKDT--LLA--DVCLAALHEGQSITQDYPKYQAQYTFSSPSQICTML A4var/3540144 VRNLENISALDKINNDT--LLA--DVCLAALHEGQSITQDYPKYQAQYASSSPSQICTML CS2-CSA/4760401 VRNLENISALDKINNDT--LLA--DVCLAALHEGQSITQDYPKYQAQYASSSPSQICTML SD106D/5817484 DRNLEQIDPAKITTTHN--LLV--DVLLAAKHEGESIIDNYPS-DHHNK---EG-ICTAL A4tres/6601435 DRNLEQIDPAKITTTHN--LLV--DVLLAAKHEGESIIDNYPS-DHHNK---EG-ICTAL K162f/2252533 DQNLEQIRPEKITSTHN--LLV--DVCQAAKFEGESIIKNYPQ-DRNNN---EV-ICTAL 3D7g9/2252550 DQNLEQIRPEQITSTDN--LLA--DVCLAAKHEGESIIKNYPQ-DRNNN---EV-ICTAL Vnm13b/2252540 DENLEVIKTENITSTDN--LLM--DVLLAAKYEGQSLMEKYPYNEHHNK---EG-ICTAL Vnm13C/2252541 DENLEVINPENITSTDN--LLM--DVLLAAKYEGQSLMEKYPYNEHHNK---EG-ICTAL Vnm13D/2252542 DENLEVINPENITSTDN--LLM--DVLLAAKYEGQSLMEKYPYSEHHNT---QS-ICPML SD126M/5817512 DRNLEHIEPDQITSTHN--LLV--DVLLAAKHEGDSIINNYPD-NRDKK---EG-ICTAL Van17/2252538 DLNLEHIDVHNVQKYFM--TLFGENVLVTAKYEGESIVEKHPNRGSSE-------VCTAL TRENDS in Parasitology

Fig. 3. Analysis of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) similarity and divergence. (a) A4var and CS2–chondroitin sulfate A (CSA) PfEMP1 diverge in sequence, structure and function at the Duffy-binding-like (DBL) 1α homology block H (Refs 22,24,27). (b) The DBLα domain with labeled homology (pink) and hypervariable (blue) blocks. Universal primer sets based on homology blocks B, D, H and J have been used to study PfEMP1 diversity in laboratory and field isolates. (c) Polymorphic tags amplified with B and D homology block primers15,44,45 or cloned var genes. The 80% consensus is based on 90 different tags. A partial list of sequences (gene name and gene identification number) is shown, and the complete alignment is available from the authors. Abbreviations: ATS, acidic terminal segment; CIDR, cysteine-rich intracellular domain region; K, Kenya; NTS, N-terminal segment; SD, Sudan; TM, transmembrane domain; Van, Vanuatu; Vnm, Vietnam. A4var and CS2–CSA sequences are colored red, with other sequences colored as variations on these.

ROSETTING but are highly variable between clones and therefore appear to be sequence dependent. A striking exception is CD36 binding, which is functionally conserved between distinct CIDR1α domains19,22,27 and is the most common property of field isolates. Although there are important exceptions that do not bind CD36, such as placental sequestered parasites28,29, the extensive conservation implies an important role in parasite survival or perhaps host

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disease outcome30–32. By extension, CD36 binding presumably had a significant, early role in PfEMP1 evolution, because it appears to be a critical interaction for sequestration in vivo17. Other adhesion domains have been mapped that are internal to the head structure. Of these, an intercellular adhesion molecule-1 (ICAM-1) binding region was shown to reside in the DBLβ–C2 tandem27 and a chondroitin sulfate A (CSA) binding region in the DBLγ type24,33. An exciting possibility is that the sequence approach might provide insights into cytoadherence and P. falciparum pathogenesis. For instance, ICAM-1 and CSA have been linked to cerebral and placental malaria, respectively. Do other PfEMP1 proteins that adhere to ICAM-1 or CSA use DBLβ–C2 and DBLγ domains? This is an important consideration for vaccine design, especially if the different DBL sequence types have slightly different structures. Moreover, if PfEMP1 proteins with particular DBL types encode special binding properties, are there more parasites expressing these variants in the severely ill? This possibility might be investigated with DBL-type-specific primers.

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Altogether, PfEMP1 proteins show a remarkable capacity to bind different molecules, apparently the result of a highly malleable structure and a byproduct of their role as variant antigens and sequestration receptors. The extreme PfEMP1 polymorphism foreshadows a large adaptive potential to evade immunity, rapidly adapt to changes in the host and acquire novel binding properties. Measuring PfEMP1 diversity

Immunochemical, functional (adhesion) and molecular biological methods are used to demonstrate and study PfEMP1 diversity. Agglutination and indirect immunofluorescence flow-cytometry studies of infected erythrocytes show that there is a high degree of diversity between isolates in neighboring and distant geographic locations, and also that there is a low degree of crossreactivity34–38. Cross-reactive natural sera have not been studied at the molecular level to show whether they react with distinct epitopes or related PfEMP1 proteins from different strains. Recently, we used monoclonal antibodies to demonstrate the existence of cross-reactive epitopes in distinct CIDR1 domains39. Although the current opinion is that natural infections induce largely variant-specific protective antibodies40, pregnant women represent a remarkable exception in that they develop strain-transcendent, CSA-adhesion-blocking antibodies that correlate with protection from disease41. Curiously, CSA-adherent strains are not agglutinated by blocking sera, in contrast to the high agglutinability of CD36-binding infected erythrocytes. A greater understanding of PfEMP1 structure and natural immunity might support the development of PfEMP1 vaccines and therapeutics. This might also enhance our understanding of P. falciparum population structure, because it has been proposed that immunity to PfEMP1 could promote the appearance of discrete strains with minimal or no PfEMP1 overlap42. Another way to assess PfEMP1 diversity is sequencing. With it taking months to identify and sequence single var genes from a laboratory isolate, and several years of intensive, multilaboratory genome sequencing to compile the 3D7 complement of var genes, measuring var gene population diversity is a daunting task. The high degree of polymorphism and the gene length (6–12 kbp) preclude full-length sequencing of an entire population’s var genes. Current technologies direct us to more modest estimates of diversity. These techniques rely on degenerate primers to the most conserved exon 1 regions, which flank regions of high polymorphism. Such PCR generates a short ‘tag’ to the polymorphic sequence for each variant. To date, the most universal primer sets have been directed to DBLα homology blocks (Fig. 3). Initial comparisons of laboratoryisolate DBLα tags showed that there is little similarity between parasite genomes. Comparing 42 http://parasites.trends.com

tags from 3D7 (Ref. 43) with 41 from A4 (ItG), the sequences range from 51% to 87% similarity (S. Kyes, unpublished). This diversity is also seen in field isolates, comparing within villages and between distant sites43–46. Although, in one study, some sequences from a village were highly similar45, there is no preference for sequences from particular geographical regions to cluster together in multiple sequence alignments. Thus, even within the head structure (the most conserved element of the PfEMP1 extracellular region), there is tremendous variation. The structural, functional or mechanistic constraints that limit variation are unknown. Although hypervariable regions diverge significantly, short amino acid runs (small hypervariable motifs) are frequently identical between PfEMP1 proteins from the same or geographically different isolates (Fig. 3)15,45. The small motifs, or closely related variations, are occasionally found shifted slightly in position between different PfEMP1 proteins (see, for example, DKIN of hypervariable region III in several PfEMP1 tags in Fig. 3). The mosaic-like relationship of DBLα hypervariable regions has been interpreted as evidence of PfEMP1 recombination15,45. The possible contribution of small hypervariable motifs to crossreactive antibody responses is unknown. Contrasting with the high degree of exon-1 polymorphism, the 5′ untranslated region of var genes is relatively conserved. One study reported two conserved classes of 5′ var (Ref. 47). Each is distinct but contains a 30 bp ‘conserved degenerate’ sequence located at different distances from the ATG start codon. One class is linked to subtelomeric var genes, whereas the other is linked to centrally located var sequences. The subtelomeric 5′ flanking sequence has also been identified in field isolates (S. Kyes, unpublished), suggesting that noncoding sequences (at least the 5′ regions) are highly conserved in evolution and are not subject to the same diversifying forces as var coding regions. The relative conservation might reflect a highly selected mechanism for tight control of expression. Studies to date indicate that 5′ flanking regions are involved in var gene expression47,48, but there are no data about whether differences affect switch rates to and from central and subtelomeric var genes. Mechanisms generating PfEMP1 diversity

The mechanisms that create var gene diversity are only starting to be revealed but recombination within these sequences is under intensive investigation. Most var genes are located within subtelomeric repeat regions4, like variant antigen genes from other protozoan parasites49. This location lends itself to a high degree of non-homologous recombination, using the subtelomeric repeat (rep20) sequences for alignment and possibly the relatively conserved var exon 2, and 5′ and 3′ var flanking sequences. Restriction analyses of genomic DNA from progeny of several genetic crosses14,15 shows

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a rate of meiotic recombination events in var genes far exceeding (at least eightfold) the expected rate50. Non-homologous recombination and gene conversion events are observed. Diversity might also be generated during mitotic recombination, as suggested by restriction analyses15. The most striking physical evidence for the possibility of large-scale non-homologous (or ectopic) recombination events is the presence of clusters of telomeres in both asexual and sexual (gametocyte) stages of the parasite, using fluorescent in situ hybridization analysis for subtelomeric repeat sequence probes14. Chromosome end clustering allows the physical alignment of similar but not identical var genes for recombination or gene conversion events. Also, in budding yeast, clustering is thought to be involved in transcriptional silencing of telomeres51. This presents the possibility that control of var gene expression might be linked to clustering of chromosome ends. Several examples of var recombination, in both the coding region7,14 and the 5′ non-coding region48, have been analyzed at the sequence level and there is now evidence that recombination can affect PfEMP1 function. A4var and CS2–CSA PfEMP1 proteins (both Ituxi strain) are nearly identical from the protein start until they diverge in sequence and structure at DBL1α homology block H (Fig. 3). The A4var binds CD36 and ICAM-1, whereas the CS2–CSA binds CSA but not CD36. Binding domains have been mapped internal to the site of divergence22,24,27. Recombination has frequently been suggested as a mechanism to promote the diversification of adhesive phenotypes. Although there needs to be more study on how PfEMP1 proteins are presented at the erythrocyte surface, the characteristic PfEMP1 protein architecture and host selection might ensure that certain domain types (head structure) always occupy a more favored position for binding. The accessibility of DBLβ, γ and δ types for binding might differ depending on PfEMP1 size or whether domains appear in the middle or at the end. Thus, recombination and domain rearrangement might silence traits, reawaken latent properties, modify affinity or leave binding unaltered simply by positional effects. The impact of recombination on immune recognition remains to be studied. Functional and antigenic sites within PfEMP1 adhesive domains

The mechanisms that create PfEMP1 diversity contribute to immune evasion but also provide opportunities to create, delete or modify existing PfEMP1 binding properties. Although there has been limited fine epitope mapping of functional and antigenic regions in PfEMP1, sequence comparisons have been useful for dissecting out conserved and variant features. These observations suggest different possible ways in which immunity and binding shape PfEMP1 structure and function. http://parasites.trends.com

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A conspicuous feature of PfEMP1 variation is length polymorphism. DBL domains display type-specific differences in length and molecular mass (Fig. 2a)12. As a rule, DBLα types are smaller than DBLδ (36.5 ± 1.2 kDa as against 46.0 ± 2.2 kDa)12. Areas of expansion are not constant between DBL domains, although certain hypervariable regions expand more than others12. Size variance might reflect another way that DBLα and δ types have specialized, perhaps owing to different structural roles. Alternatively, binding constraints might be tightened for α types and relaxed for δ types. When the DBL structure is solved it will be interesting to learn how the ‘accordion-like’ length expansion is accommodated. By comparison, DBL domains of EBA175 did not differ in length between strains, although point mutations were detected52, a possible consequence of their being a less immunodominant target. Functional sites within PfEMP1 DBL domains have not been reported, although binding sites within EBA DBL domains have been identified. Critical binding residues for both the Plasmodium vivax EBA and P. knowlesi β EBA map between cysteines 4 and 7 even though they recognize distinct erythrocyte surface molecules53. It will be interesting to investigate whether other DBL adhesive traits map to this region and the extent to which it includes a general binding area whose specificity depends upon sequence. A critical binding region has also been mapped within the CIDR domain. In the Malayan Camp var1 CIDR1α, the minimal CD36 binding site was localized to a central fragment19. The analogous region from other PfEMP1 proteins also bound CD36 (Ref. 19), with small amino acid changes such as DIE to GHR responsible for loss or gain of function54. The minimal CIDR binding region was named M2 and the flanking regions M1 and M3 (Fig. 2b). Within the M2, the third cysteine in the cysteine-rich motif (Fig. 2b) was shown to be critical for Malayan Camp var1 CIDR1α binding to CD36 (Ref. 19). This cysteine is not present in many CIDRβ type domains and might preclude their binding CD36. CIDR domains are remarkable in that they maintain CD36 binding despite tremendous amino acid variation. Polymorphism includes the M1 and M2 areas but is especially prominent in the M3 region, which is not conserved and frequently contains long runs of charged residues (Fig. 2b)12. The M3 area might divert antibody from functional regions. Currently, there is considerable interest in testing whether cross-reactive antibody responses can be raised based upon the minimal CIDR binding region. Humans infected with P. falciparum develop strain-specific CD36 adhesion blocking antibodies55, but neither pooled human nor monkey immune sera reacted with a M2 recombinant protein19. Thus, it will be important to investigate whether the CIDR minimal binding site is poorly recognized during natural infections. Mapping the specificity of

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adhesion-blocking antibodies might offer insights to focus vaccine-directed immunity on conserved and critical PfEMP1 sites. Conclusions

Evidence has now accumulated to establish that PfEMP1 have an essential role in P. falciparum cytoadherence. The dual responsibility of PfEMP1 as References 1 Baruch, D.I. (1999) Adhesive receptors on malaria-parasitized red cells. Baillieres Best Pract. Res. Clin. Haematol. 12, 747–761 2 Cooke, B. et al. (2000) Falciparum malaria: sticking up, standing out and out-standing. Parasitol. Today 16, 416–420 3 Su, X.Z. et al. (1995) The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82, 89–100 4 Rubio, J.P. et al. (1996) The var genes of Plasmodium falciparum are located in the subtelomeric region of most chromosomes. EMBO J. 15, 4069–4077 5 Fischer, K. et al. (1997) Expression of var genes located within polymorphic subtelomeric domains of Plasmodium falciparum chromosomes. Mol. Cell Biol. 17, 3679–3686 6 Thompson, J.K. et al. (1997) The chromosomal organization of the Plasmodium falciparum var gene family is conserved. Mol. Biochem. Parasitol. 87, 49–60 7 Baruch, D.I. et al. (1995) Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82, 77–87 8 Smith, J.D. et al. (1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82, 101–110 9 Adams, J.H. et al. (1990) The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell 63, 141–153 10 Chitnis, C.E. and Miller, L.H. (1994) Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion. J. Exp. Med. 180, 497–506 11 Sim, B.K. et al. (1994) Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum. Science 264, 1941–1944 12 Smith, J.D. et al. (2000) Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family. Mol. Biochem. Parasitol. 110, 293–310 13 Adams, J.H. et al. (1992) A family of erythrocyte binding proteins of malaria parasites. Proc. Natl. Acad. Sci. U. S. A. 89, 7085–7089 14 Freitas-Junior, L.H. et al. (2000) Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum. Nature 407, 1018–1022 15 Taylor, H.M. et al. (2000) var gene diversity in Plasmodium falciparum is generated by frequent recombination events. Mol. Biochem. Parasitol. 110, 391–397 16 McCormick, C.J. et al. (1997) Intercellular adhesion molecule-1 and CD36 synergize to mediate adherence of Plasmodium falciparumhttp://parasites.trends.com

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variant antigen and binding ligand has produced a highly diverse protein family whose regulation is still incompletely understood. Sequence analysis has revealed conserved and variant PfEMP1 features. The sequence approach provides insight into PfEMP1 structure and function with potentially significant implications for malaria disease and vaccine and therapeutic interventions.

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31 Pain, A. et al. (2001) Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria. Proc. Natl. Acad. Sci. U. S. A. 98, 1805–1810 32 Pain, A. et al. (2001) A non-sense mutation in Cd36 gene is associated with protection from severe malaria. Lancet 357, 1502–1503 33 Buffet, P.A. et al. (1999) Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: a receptor for human placental infection. Proc. Natl. Acad. Sci. U. S. A. 96, 12743–12748 34 Marsh, K. and Howard, R.J. (1986) Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants. Science 231, 150–153 35 Forsyth, K.P. et al. (1989) Diversity of antigens expressed on the surface of erythrocytes infected with mature Plasmodium falciparum parasites in Papua New Guinea. Am. J. Trop. Med. Hyg. 41, 259–265 36 Aguiar, J.C. et al. (1992) Agglutination of Plasmodium falciparum-infected erythrocytes from east and west African isolates by human sera from distant geographic regions. Am. J. Trop. Med. Hyg. 47, 621–632 37 Bull, P.C. et al. (1999) Antibody recognition of Plasmodium falciparum erythrocyte surface antigens in Kenya: evidence for rare and prevalent variants. Infect. Immun. 67, 733–739 38 Giha, H.A. et al. (1999) Overlapping antigenic repertoires of variant antigens expressed on the surface of erythrocytes infected by Plasmodium falciparum. Parasitology 119, 7–17 39 Gamain, B. et al. (2001) The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes. Proc. Natl. Acad. Sci. U. S. A. 98, 2664–2669 40 Bull, P.C. et al. (1998) Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat. Med. 4, 358–360 41 Fried, M. et al. (1998) Maternal antibodies block malaria. Nature 395, 851–852 42 Gupta, S. et al. (1998) Chaos, persistence, and evolution of strain structure in antigenically diverse infectious agents. Science 280, 912–915 43 Taylor, H.M. et al. (2000) A study of var gene transcription in vitro using universal var gene primers. Mol. Biochem. Parasitol. 105, 13–23 44 Kyes, S. et al. (1997) Genomic representation of var gene sequences in Plasmodium falciparum field isolates from different geographic regions. Mol. Biochem. Parasitol. 87, 235–238 45 Ward, C.P. et al. (1999) Analysis of Plasmodium falciparum PfEMP-1/var genes suggests that recombination rearranges constrained sequences. Mol. Biochem. Parasitol. 102, 167–177 46 Kirchgatter, K. et al. (2000) Plasmodium falciparum: DBL-1 var sequence analysis in field isolates from central Brazil. Exp. Parasitol. 95, 154–157

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Fc receptors and immunity to parasites Richard J. Pleass and Jenny M. Woof Fc receptors (FcRs) are crucial in the immune system; they mediate a plethora of biological functions as diverse as antigen presentation, phagocytosis, cytotoxicity, induction of inflammatory cascades and modulation of immune responses. Parasites, in order to survive in the immunocompetent host, have devised ingenious methods to subvert this important aspect of the immune response. This article discusses the current thinking on FcRs, their role in immunity to parasites, and immune evasion strategies employed by parasites in their attempt to neutralize the important immune defense mechanisms mediated by these molecules.

Richard J. Pleass* Jenny M. Woof Dept. of Molecular and Cellular Pathology, University of Dundee Medical School, Ninewells Hospital, Dundee, UK DD1 9SY. *e-mail: [email protected]

Most immunoglobulin receptors have evolved as part of the immunoglobulin gene superfamily (IgSF)1. Important examples from this family include receptors for IgG (FcγRI, FcγRII, FcγRIII), IgE (FcεRI), IgA [FcαRI, Fcα/µR, polymeric immunoglobulin receptor (pIgR)] and IgM (Fcα/µR) (Table 1). By contrast, the low-affinity IgE receptor (FcεRII, CD23) is a C-type lectin. Most genes encoding FcRs map to human chromosome 1q32.3, while curiously the FcαRI gene lies on chromosome 19q13.4 and shares homology with adjacent genes, including those encoding natural killer cell inhibitory receptors (KIRs) and the Ig-like transcripts (ILTs). The IgSF FcRs each possess a unique ligandbinding chain (α-subunit) with a transmembrane region which is often complexed with more promiscuous signaling chains (β and γ subunits; Fig. 1). A notable exception is FcγRIIIb, which is linked to the lipid bilayer of neutrophils by a glycophosphatidylinositol (GPI) anchor. The effector mechanisms of the receptors are triggered through sequences in the intracellular region of their subunit http://parasites.trends.com

chains. These signaling motifs are designated immunoreceptor tyrosine-based activation or inhibitory motifs (ITAMs or ITIMs). The motifs bear characteristic tyrosine-containing sequences, typically YxxL(x7)YxxL (ITAM) or I/VxYxxL/V (ITIM), which, when phosphorylated following receptor crosslinking, serve as sites either promoting (ITAM) or negatively regulating (ITIM) activation of cytoplasmic proteins into signaling complexes2 (Table 1). The structural diversity of the Fc receptor family and their broad distribution on different cells of the immune system provides for a wide range of functional roles1 (Table 1). FcR polymorphisms and susceptibility to infection

Particular allelic polymorphisms in FcγRIIa and FcγRIII are associated with differential susceptibility to certain infections. For example, one FcγRIIa allele (FcγRIIa-Arg131) is associated with increased susceptibility to infection by encapsulated bacteria, such as Haemophilus influenzae, Streptococcus pneumoniae and Neiserria meningitidis, which elicit IgG2 responses3. This allele cannot bind IgG2 and is therefore unable to elicit clearance of IgG2-coated bacteria. Associations of FcR polymorphisms with susceptibility to protozoan or worm infections are only beginning to be investigated. Kenyan infants homozygous for the FcγRIIa-Arg131 allele are reported to be less at risk from high-density Plasmodium falciparum infection compared with children with the heterozygous Arg/His131

1471-4922/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)02086-4