Blood Stage Research Articles

Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5

There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.

Biogenically synthesized green silver nanoparticles exhibit antimalarial activity

The suboptimal efficacies of existing anti-malarial drugs attributed to the emergence of drug resistance dampen the clinical outcomes. Hence, there is a need for developing novel drug and drug targets. Recently silver nanoparticles (AgNPs) constructed with the leaf extracts of Euphorbia cotinifolia were shown to possess antimalarial activity. Therefore, the synthesized AgNPs from Euphorbia cotinifolia (EcAgNPs) were tested for their parasite clearance activity. We determined the antimalarial activity in the asexual blood stage infection of 3D7 (laboratory strain) P. falciparum. EcAgNPs demonstrated the significant inhibition of parasite growth (EC50 of 0.75 µg/ml) in the routine in vitro culture of P. falciparum. The synthesized silver nanoparticles were seen to induce apoptosis in P. falciparum through increased reactive oxygen species (ROS) ROS production and activated programmed cell death pathways characterized by the caspase-3 and calpain activity. Also, altered transcriptional regulation of Bax/Bcl-2 ratio indicated the enhanced apoptosis. Moreover, inhibited expression of PfLPL-1 by EcAgNPs is suggestive of the dysregulated host fatty acid flux via parasite lipid storage. Overall, our findings suggest that EcAgNPs are a non-toxic and targeted antimalarial treatment, and could be a promising therapeutic approach for clearing malaria infection.

Progressive heterogeneity of enlarged and irregularly shaped apicoplasts in P. falciparum persister blood stages after drug treatment.

Morphological modifications and shifts in organelle relationships are hallmarks of dormancy in eukaryotic cells. Communications between altered mitochondria and nuclei are associated with metabolic quiescence of cancer cells that can survive chemotherapy. In plants, changes in the pathways between nuclei, mitochondria, and chloroplasts are associated with cold stress and bud dormancy. Plasmodium falciparum parasites, the deadliest agent of malaria in humans, contain a chloroplast-like organelle (apicoplast) derived from an ancient photosynthetic symbiont. Antimalarial treatments can fail because a small fraction of the blood stage parasites enter dormancy and recrudesce after drug exposure. Altered mitochondrial-nuclear interactions in these persisters have been described for P. falciparum, but interactions of the apicoplast remained to be characterized. In the present study, we examined the apicoplasts of persisters obtained after exposure to dihydroartemisinin (a first-line antimalarial drug) followed by sorbitol treatment, or after exposure to sorbitol treatment alone. As previously observed, the mitochondrion of persisters was consistently enlarged and in close association with the nucleus. In contrast, the apicoplast varied from compact and oblate, like those of active ring stage parasites, to enlarged and irregularly shaped. Enlarged apicoplasts became more prevalent later in dormancy, but regular size apicoplasts subsequently predominated in actively replicating recrudescent parasites. All three organelles, nucleus, mitochondrion, and apicoplast, became closer during dormancy. Understanding their relationships in erythrocytic-stage persisters may lead to new strategies to prevent recrudescences and protect the future of malaria chemotherapy.

Hyper-diverse antigenic variation and resilience to transmission-reducing intervention in falciparum malaria

Intervention efforts against falciparum malaria in high-transmission regions remain challenging, with rapid resurgence typically following their relaxation. Such resilience co-occurs with incomplete immunity and a large transmission reservoir from high asymptomatic prevalence. Incomplete immunity relates to the large antigenic variation of the parasite, with the major surface antigen of the blood stage of infection encoded by the multigene and recombinant family known as var. With a stochastic agent-based model, we investigate the existence of a sharp transition in resurgence ability with intervention intensity and identify molecular indicators informative of its proximity. Their application to survey data with deep sampling of var sequences from individual isolates in northern Ghana suggests that the transmission system was brought close to transition by intervention with indoor residual spraying. These results indicate that sustaining and intensifying intervention would have pushed malaria dynamics to a slow-rebound regime with an increased probability of local parasite extinction.

Plasmodium falciparum Mitochondrial Complex III, the Target of Atovaquone, Is Essential for Progression to the Transmissible Sexual Stages.

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

A broadly cross-reactive i-body to AMA1 potently inhibits blood and liver stages of Plasmodium parasites

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages.

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. We isolated 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. HumAb 826827 blocked the invasion of human erythrocytes using Pv clinical isolates and inhibited sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 to 3.7 ug/mL). It also significantly reduced liver infection of chimeric FRG humHep mice in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate transcendent, blocks both pre erythrocytic and blood stage infection, and could be a new therapy for Pv.

Cell Death of P. vivax Blood Stages Occurs in Absence of Classical Apoptotic Events and Induces Eryptosis of Parasitized Host Cells.

Elucidation of pathways regulating parasite cell death is believed to contribute to identification of novel therapeutic targets for protozoan diseases, and in this context, apoptosis-like cell death has been reported in different groups of protozoa, in which metacaspases seem to play a role. In the genus Plasmodium, apoptotic markers have been detected in P. falciparum and P. berghei, and no study focusing on P. vivax cell death has been reported so far. In the present study, we investigated the susceptibility of P. vivax to undergo apoptotic cell death after incubating mature trophozoites with the classical apoptosis inducer staurosporine. As assessed by flow cytometry assays, staurosporine inhibited parasite intraerythrocytic development, which was accompanied by a decrease in cell viability, evidenced by reduced plasmodial mitochondrial activity. However, typical signs of apoptosis, such as DNA fragmentation, chromatin condensation, and nuclear segregation, were not detected in the parasites induced to cell death, and no significant alteration in metacaspase gene expression (PvMCA1) was observed under cell death stimulus. Interestingly, dying parasites positively modulated cell death (eryptosis) of host erythrocytes, which was marked by externalization of phosphatidylserine and cell shrinkage. Our study shows for the time that P. vivax blood stages may not be susceptible to apoptosis-like processes, while they could trigger eryptosis of parasitized cells by undergoing cell death. Further studies are required to elucidate the cellular machinery involved in cell death of P. vivax parasites as well as in the modulation of host cell death.

Experimental vaccination by single dose sporozoite injection of blood-stage attenuated malaria parasites

Malaria vaccination approaches using live Plasmodium parasites are currently explored, with either attenuated mosquito-derived sporozoites or attenuated blood-stage parasites. Both approaches would profit from the availability of attenuated and avirulent parasites with a reduced blood-stage multiplication rate. Here we screened gene-deletion mutants of the rodent parasite P. berghei and the human parasite P. falciparum for slow growth. Furthermore, we tested the P. berghei mutants for avirulence and resolving blood-stage infections, while preserving sporozoite formation and liver infection. Targeting 51 genes yielded 18 P. berghei gene-deletion mutants with several mutants causing mild infections. Infections with the two most attenuated mutants either by blood stages or by sporozoites were cleared by the immune response. Immunization of mice led to protection from disease after challenge with wild-type sporozoites. Two of six generated P. falciparum gene-deletion mutants showed a slow growth rate. Slow-growing, avirulent P. falciparum mutants will constitute valuable tools to inform on the induction of immune responses and will aid in developing new as well as safeguarding existing attenuated parasite vaccines.

Antiplasmodial action of 4-nitrobenzenesulfonamide chalcones: Design, synthesis, characterisation, in vitro and in silico evaluation against blood stages of Plasmodium falciparum 3D7.

Malaria is an intracellular protozoan parasitic disease caused by Plasmodium species with significant morbidity and mortality in endemic regions. The complex lifecycle of the parasite and the emergence of drug-resistant Plasmodium falciparum have hampered the efficacy of current anti-malarial agents. To circumvent this situation, the present study attempts to demonstrate the blood-stage anti-plasmodial action of 26 hybrid compounds containing the three privileged bioactive scaffolds (sulfonamide, chalcone, and nitro group) with synergistic and multitarget action. These three parent scaffolds exhibit divergent activities, such as antibacterial, anti-malarial, anti-fungal, anti-inflammatory, and anticancer. All the synthesised compounds were characterised using various spectroscopic techniques. The in vitro blood-stage inhibitory activity of 26 hybrid compounds was evaluated against mixed-stage culture (asynchronize) of human malarial parasite P. falciparum, Pf 3D7 at different concentrations ranging from 25.0 µg/mL to 0.78 µg/mL using SYBR 1 green assay, with IC50 values determined after 48 h of treatment based on the drug-response curves. Two potent compounds (11 and 10), with 2-Br and 2,6-diCl substitutions, showed pronounced activity with IC50 values of 5.4 µg/mL and 5.6 µg/mL, whereas others displayed varied activity with IC50 values ranging from 7.0 µg/mL to 22.0 µg/mL. Both 11 and 10 showed greater susceptibility towards mature-stage trophozoites than ring-stage parasites. The hemolytic and in vitro cytotoxicity assays revealed that compounds 11 and 10 did not cause any toxic effects on host red blood cells (uninfected), human-derived Mo7e cells, and murine-derived BA/F3 cells. The in vitro observations are consistent with the in silico studies using P. falciparum-dihydrofolate reductase, where 11 and 10 showed a binding affinity of -10.4 Kcal/mol. This is the first report of the hybrid scaffold, 4-nitrobenzenesulfonamide chalcones, demonstrating its potential as an anti-plasmodial agent.

Mass Spectrometric and Artificial Intelligence-Based Identification of the Secretome of Plasmodium falciparum Merozoites to Provide Novel Candidates for Vaccine Development Pipeline.

Merozoites are the only extracellular form of blood stage parasites, making it a worthwhile target. Multiple invasins that are stored in the merozoite apical organelles, are secreted just prior to invasion, and mediates its interaction with RBC. A comprehensive identification of all these secreted invasins is lacking and this study addresses that gap. Pf3D7 merozoites were enriched and triggered to discharge apical organelle contents by exposure to ionic conditions mimicking that of blood plasma. The secreted proteins were separated from cellular contents and both the fractions were subjected to proteomic analysis. Also, the identified secreted proteins were subjected to GO, PPI network analysis, and AI-based in silico approach to understand their vaccine candidacy. A total of 63 proteins were identified in the secretory fraction with membrane and apical organellar localization. This includes various MSPs, micronemal EBAs and rhoptry bulb proteins, which play a crucial role in initial and late merozoite attachment, and majority of them qualified as vaccine candidates. We, for the first time, report the secretory repertoire of merozoite and its status for vaccine candidacy. This information can be utilized to develop better invasion blocking multisubunit vaccines, comprising of immunological epitopes from several secreted invasins.

Additional blood meals increase sporozoite infection in Anopheles mosquitoes but not Plasmodium falciparum genetic diversity

The availability of nutrients from mosquito blood meals accelerates the development of Plasmodium falciparum laboratory strains in artificially infected Anopheles gambiae mosquitoes. The impact of multiple blood meals on the number of P. falciparum genotypes developing from polyclonal natural human malaria infections (field-isolates) remains unexplored. Here, we experimentally infect An. gambiae with P. falciparum field-isolates and measure the impact of an additional non-infectious blood meal on parasite development. We also assess parasite genetic diversity at the blood stage level of the parasite in the human host and of the sporozoites in the mosquito. Additional blood meals increase the sporozoite infection prevalence and intensity, but do not substantially affect the genetic diversity of sporozoites in the mosquito. The most abundant parasite genotypes in the human blood were transmitted to mosquitoes, suggesting that there was no preferential selection of specific genotypes. This study underlines the importance of additional mosquito blood meals for the development of parasite field-isolates in the mosquito host.

Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P.falciparum parasite challenge invivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Diversity and selection analyses identify transmission-blocking antigens as the optimal vaccine candidates in Plasmodium falciparum

A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum's life cycle. We analysed 325P.falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Database. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis. Among the ten antigens analysed, only three in the transmission-blocking vaccine category display P. falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP119 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5, a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS, the transmission-blocking antigens Pfs25, Pfs48/45, Pfs230, Pfs47, and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission. These findings offer valuable insights into the selection of optimal vaccine candidates against P.falciparum. Based on our results, we recommend prioritising conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives. Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680).

Optimization of pyrazolopyridine 4-carboxamides with potent antimalarial activity for which resistance is associated with the P. falciparum transporter ABCI3

Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7′-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class.

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

Disruption of Plasmodium falciparum kinetochore proteins destabilises the nexus between the centrosome equivalent and the mitotic apparatus

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.

Hepatotoxic Effects of an Oral Amiodarone in White Albino Rats: Sub-Acute Biochemical, and Histopathological Assessments

Amiodarone, potent antidysrhythmic, widely used drug that has been associated with hepatic toxicity in long-term or excessive use. In the presented study twelve rats were allocated into two groups and given daily doses via gastric gavage orally for two weeks as follows; The first group served as a control normal group, whereas the second got amiodarone at a dosage of 300mg/kg/day orally. Liver tissues were processed for light microscopy, and blood samples were examined for serum transaminases (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and phospholipase a2 enzyme (Pla2) (which are thought to be an indicator of phospholipidosis and lipid buildup). Amiodarone was shown to produce hepatic histological abnormalities such as blood vessel congestion, leucocytic infiltration, liver degeneration, and stages of steatosis and necrosis of hepatocytes. The biochemical assessments were revealed that there was an elevation in amiodarone group's ALT and AST levels as a result of hepatic necrosis leading to leakage of enzymes content, while serum Pla2 was lowered significantly. Also, histopathology indicates stages of steatosis (Lipid accumulation) in hepatocytes, which may lead to farther damage in the late stages of hepatotoxicity.

Comparative proteomics uncovers low asparagine content in Plasmodium tRip-KO proteins.

tRNAs are not only essential for decoding the genetic code, but their abundance also has a strong impact on the rate of protein production, folding, and on the stability of the translated messenger RNAs. Plasmodium expresses a unique surface protein called tRip, involved in the import of exogenous tRNAs into the parasite. Comparative proteomic analysis of the blood stage of wild-type and tRip-KO variant of P. berghei parasites revealed that downregulated proteins in the mutant parasite are distinguished by a bias in their asparagine content. Furthermore, the demonstration of the possibility of charging host tRNAs with Plasmodium aminoacyl-tRNA synthetases led us to propose that imported host tRNAs participate in parasite protein synthesis. These results also suggest a novel mechanism of translational control in which import of host tRNAs emerge as regulators of gene expression in the Plasmodium developmental cycle and pathogenesis, by enabling the synthesis of asparagine-rich regulatory proteins that efficiently and selectively control the parasite infectivity.

Target-agnostic identification of human antibodies to Plasmodium falciparum sexual forms reveals cross stage recognition of glutamate-rich repeats.

A naturally acquired human monoclonal antibody recognizes proteins expressed at different stages of the Plasmodium falciparum lifecycle through affinity-matured homotypic interactions with glutamate-rich repeats.