Figure - available from: Glycoconjugate Journal
This content is subject to copyright. Terms and conditions apply.
Kinetic analysis of rVAR2 interactions with CS10k species using SPR. KD (a) and Rmax (b) values from the analysis of rVAR2 interacting with different sulfated 10 k CS species were determined and plotted against D4S. Data are shown as the average ± S.D. of three independent experiments
Source publication
Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placent...
Citations
... Asimismo, la infección que ocurre en las etapas tempranas del embarazo, durante el primer trimestre, ocasiona restricción del crecimiento intrauterino en las etapas tardías del mismo, produciendo una disminución del peso de la placenta y, por ende, de su desarrollo vascular, en cualquiera de los tres procesos de vasculogénesis, angiogénesis ramificante o no ramificante, provocando inhibición en la migración trofoblástica uterina, disminución en el volumen de sangre transportada por los espacios intervellosos; lo que se manifiesta clínicamente en el segundo y en el tercer trimestre de gestación. Ahora bien, si la infección ocurre en las etapas tardías del embarazo, la vascularización placentaria tiende a incrementarse por procesos compensatorios que tienden a proteger a la madre y al feto de la disminución del transporte de oxígeno y nutrientes, aunque esto podría desencadenar en la generación de una placenta aumentada de tamaño (31,34,42). ...
... También se ha evidenciado que bajos niveles de IL-5 se asocian a mayor riesgo de presentar partos pretérmino y neonatos pequeños para su edad gestacional (43,45,46). Esta tormenta de citoquinas provoca desórdenes en el transporte de aminoácidos y glucosa, así como en la expresión y distribución de las moléculas transportadoras de nutrientes y hormonas de crecimiento (34,42). ...
Malaria infections increase the risk of complications in the mother-fetus binomial. In Venezuela, the casuistry of this vulnerable group has not been updated in recent years. The objective of this narrative review was to comprehensively describe what pregnancy-associated malaria is and its maternal, fetal and neonatal effects; trying to answer the following research question. All forms of pregnancy-associated malaria, including gestational, placental and congenital malaria, cause maternal-fetal and neonatal alterations that, if they progress, could lead to the death of this binomial. Physiopathology and immunopathology can explain the symptoms of pregnant women and the fetus, as well as their complications; depending on the parasitic form affecting. There are new updates in the diagnosis, prevention and treatment of this entity.
... 31 The infectious mechanisms of the parasite, as well as the adverse effects and maternal-fetal complications associated with the infection, are mediated by a variant of the erythrocyte membrane protein family 1 (PfEMP1), called VAR2CSA, -an antigen with a molecular weight of 350 kDa, expressed on the surface of infected erythrocytes -, which binds to its membrane receptor, i.e., chondroitin sulfate A, -a syndecane 1 (CD138)-bound glycosaminoglycan in the syncytotrophoblasts of the intervillious spaces of placental tissue. VAR2CSA has multiple regions of Duffy Binding-like (DBL) domains and interdomain-like domains, out of which, DBL 2, 3, 5 and 6 are those that bind to chondroitin sulfate A. This binding induces migration from the peripheral circulation of parasitized erythrocytes into the host tissue, a process called erythrocyte sequestration, 9,10,[33][34][35][36][37][38][39][40][41][42] and the release of bioactive molecules by the parasite, including glycosylphosphatidylinositol, stimulates maternal mononuclear cells. 43 All this occurs in three clearly demarcated phases: the activeacute phase, being the one where there is a minimal presence of placental parasites in fibrin pigment deposits; in the active-chronic phase, the presence of such parasites increases; and in the last phase, where pigments do not present parasites. ...
... Nonetheless, if infection occurs in the late stages of pregnancy, placental vascularization tends to be increased by compensatory processes that tend to protect the mother and the fetus from reduced oxygen and nutrient transport, although this could trigger the generation of an enlarged placenta. 31,34,42 In addition, malarial infection in pregnancy directly affects the biogenesis pathway of L-arginine and nitric oxide, which could also explain what has been described so far. L-arginine is the immediate precursor of nitric oxide synthase in the genesis of nitric oxide, whose bioavailability levels are necessary to regulate the previously mentioned pro-angiogenic factors that allow adequate placental vascularization, by trophoblastic expression of VEGF and PIGF, and, in that way, decreasing anti-angiogenic factors. ...
... 43,45,46 This cytokine storm causes disorders in the transport of amino acids and glucose, as well as in the expression and distribution of the molecules transporting nutrients and growth hormones. 34,42 Malarial infection in pregnancy also activates the complement system, at a systemic level and in the mother-fetus interface, with excessive increase of C1q, C3d, C4, C9, C3aR and C5aR, inducing a proinflammatory state in said interface by synergistic induction of the inflammatory cascade associated with the complement. This also results in deregulation of pro-angiogenic factors, including VEGF, sFlT-1 and angiopoyetins 1 and 2, as well as impairment in fetal neurodevelopment, preventing optimization of the fetus' intrinsic neuronal complex. ...
Aim: to review and describe exhaustively the implications of malaria in pregnancy, including its maternal, fetal, and neonatal clinical manifestations and effects; immunopathology and pathophysiology; advancements in its diagnostics, histopathology, and treatment options; and epidemiology, particularly in Venezuela, a country where its data is almost non-existent. Methods: the information used to write this manuscript was obtained during a three-month period, between June and September 2022, from specialized literature, written in English and Spanish, related to malaria associated with pregnancy, mainly published during the last five years, using journals found in the most relevant medical digital archives, including PubMed, SciELO, Elsevier, Google Scholar, Latindex, and Cochrane Plus. Among the keywords used for obtaining this updated information were malaria; malaria in pregnancy; gestational malaria; placental malaria; congenital malaria. Results: all the clinical forms related to malaria in pregnancy, including gestational, placental, and congenital malaria, can cause maternal-fetal alterations, that, in case of progressing, could lead to the death of this binomial. Their pathophysiology and immunopathology can explain the gestational and fetal symptomatology, as well as their complications, depending on the parasite form that affected them. There are new updates regarding the diagnostics, prevention, and treatment of this medical entity. Conclusion: it is imperative to exalt the relevance of studying this disease in pregnant patients, especially in the Venezuelan topography, a focus of infection with a plethora of cases of said entity, whose lack of updated epidemiological data, regarding its prevalence and incidence, is profoundly preoccupying. Pregnant patients are not only one of the most vulnerable risk groups of this parasitosis, but also have the capacity of duplicating the risk of infecting the fetus.
... It might be more effective to obtain CS with a homogeneous structure, because the interaction between CS and proteins depends on the specific structures of the molecules. Sugiura et al. have demonstrated that homogeneous 20 mers of CSA showed the highest interaction with the placental malaria protein VAR2CSA [6]. Taking these factors into consideration, we aimed to develop a method for the preparation of homogeneous non-animal-derived CSA. ...
... In the former approach, transglycosylation reactions require a transition state analog of CSA disaccharide as a substrate, the preparation process of which is very complicated. Thus, the latter approach has been selected and considered [6,13,14]. ...
Chondroitin 4-O-sulfotransferase-1 (C4ST-1) is a key enzyme for the biosynthesis of chondroitin sulfate A (CSA). Several past reports have evaluated the expression of C4ST-1 in bacteria. Since N-glycosylation is critical for the activity of C4ST-1, its activity was quite low. Here, we established a method for inducing a high expression of recombinant C4ST-1 in Escherichia coli (E. coli) by using a trigger factor (TF) fusion system. This approach enabled the expression of 18.9 ± 5.0 mg/L of TF-fused C4ST-1 (TF-C4ST-1). Although TF-C4ST-1 does not undergo N-glycosylation, the kcat/Km was approximately 60% of that of N-glycosylated C4ST-1. By using TF-C4ST-1 and chondroitin (CH) obtained from the culture of another recombinant E. coli, we succeeded in the 10-mg-scale preparation of CSA consisting almost entirely of the CSA disaccharide, GlcAβ1-3GalNAc(4 S). These results demonstrated that the expression of C4ST-1 in bacteria is sufficient to achieve the long-term goal of manufacturing non-animal-derived CSA.
... Then, two mutated recombinant KfoC enzymes were expressed each being selective for one of the substrates (UDP-GlcA or UDP-GalNAc) and the engineered enzymes, immobilized in beads, were used to elongate the chondroitin chain. The same group employed chemoenzymatic strategies for the preparation of CS oligosaccharides and polysaccharides with different sulfation patterns using the CS polymerase KfoC and recombinant sulfotransferases C4OST, C6OST, GalNAc4S-6OST and 2OST [153,154,155]. The chondroitin backbone was obtained by desulfating commercial CS from different sources and HEK293T cells were used to express the human sulfotransferases. ...
Chondroitin sulfate (CS) is a glycosaminoglycan with a broad range of applications being a popular dietary supplement for osteoarthritis. Usually, CS is extracted from animal sources. However, the known risks of animal products use have been driving the search for alternative methods and sources to obtain this compound. Several pathogenic bacteria naturally produce chondroitin-like polysaccharides through well-known pathways and, therefore, have been the basis for numerous studies that aim to produce chondroitin using non-pathogenic hosts. However, the yields obtained are not enough to meet the high demand for this glycosaminoglycan. Metabolic engineering strategies have been used to construct improved heterologous hosts. The identification of metabolic bottlenecks and regulation points, and the screening for efficient enzymes are key points for constructing microbial cell factories with improved chondroitin yields to achieve industrial CS production. The recent advances on enzymatic and microbial strategies to produce non-animal chondroitin are herein reviewed. Challenges and prospects for future research are also discussed.
... The absolute requirement of continuous CS stretches enriched in 4-O-sulfated N-acetylgalactosamine units in VAR2CSA binding have been reported in multiple studies using CS substrates derived from animal sources, as well as synthetic material (4,15,(19)(20)(21). Initial findings suggested that the minimal length of the CS binding motif is a dodecasaccharide (dp12) containing a minimum of 2-3 (19) or 4-5 (22) 4-O-sulfated Nacetylgalactosamine units. ...
... Initial findings suggested that the minimal length of the CS binding motif is a dodecasaccharide (dp12) containing a minimum of 2-3 (19) or 4-5 (22) 4-O-sulfated Nacetylgalactosamine units. More recently we have confirmed the minimal requirement of a dp12, although longer oligosaccharides showed higher affinity binding (21). Contrary to early results suggesting a requirement for several non-sulfated sugars (19), we have shown that oligosaccharides with a high degree of 4-O-sulfation are more potent in inhibiting binding of rVAR2 to isolated CSPGs compared to those that were mostly nonsulfated (21). ...
... More recently we have confirmed the minimal requirement of a dp12, although longer oligosaccharides showed higher affinity binding (21). Contrary to early results suggesting a requirement for several non-sulfated sugars (19), we have shown that oligosaccharides with a high degree of 4-O-sulfation are more potent in inhibiting binding of rVAR2 to isolated CSPGs compared to those that were mostly nonsulfated (21). To expand this to cellular binding we deconstructed the CS biosynthesis pathway by creating a library of enzyme knockouts using CRISPR/Cas9 in CHO cells (23). ...
Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant sub-fragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80-85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.
... The parasite induces expression of the malarial protein VAR2CSA on the surface of infected erythrocytes, which enables their binding to structural variants of CS in the placental intervillous space of pregnant women (Kane and Taylor-Robinson, 2011). This interaction is dependent on the size and sulfation-type of the polysaccharide, as longer chains with higher degree of C4-O-sulfation of GalNAc residues display stronger binding (Sugiura et al., 2016;Ma et al., 2021). A VAR2CSA-affinity column was recently used to enrich for CSPGs, capable of binding the VAR2CSA-protein, in human placenta. ...
Chondroitin sulfate proteoglycans (CSPGs) are found at cell surfaces and in connective tissues, where they interact with a multitude of proteins involved in various pathophysiological processes. From a methodological perspective, the identification of CSPGs is challenging, as the identification requires the combined sequencing of specific core proteins, together with the characterization of the CS polysaccharide modification(s). According to the current notion of CSPGs, they are often considered in relation to a functional role in which a given proteoglycan regulates a specific function in cellular physiology. Recent advances in glycoproteomic methods have, however, enabled the identification of numerous novel chondroitin sulfate core proteins, and their glycosaminoglycan attachment sites, in humans and in various animal models. In addition, these methods have revealed unexpected structural complexity even in the linkage regions. These findings indicate that the number and structural complexity of CSPGs are much greater than previously perceived. In light of these findings, the prospect of finding additional CSPGs, using improved methods for structural and functional characterizations, and studying novel sample matrices in humans and in animal models is discussed. Further, as many of the novel CSPGs are found in low abundance and with not yet assigned functions, these findings may challenge the traditional notion of defining proteoglycans. Therefore, the concept of proteoglycans is considered, discussing whether “a proteoglycan” should be defined mainly on the basis of an assigned function or on the structural evidence of its existence.
... As CSPGs are also present within the microvascular system, notably in the lungs and brain (53), the reason for exclusive placental sequestration of VAR2CSAexpressing IEs remains unclear. A body of work elucidated some comprehensive elements by demonstrating that the interaction of VAR2CSA with CSA is highly correlated with the degree of C-4 sulfation and the length of the CS chain (54)(55)(56), which may vary in different tissues. CSA density and wall shear stress also appear as two components influencing the IEs binding to CSA (57). ...
Over 30 million women living in P. falciparum endemic areas are at risk of developing malaria during pregnancy every year. Placental malaria is characterized by massive accumulation of infected erythrocytes in the intervillous space of the placenta, accompanied by infiltration of immune cells, particularly monocytes. The consequent local inflammation and the obstruction of the maternofetal exchanges can lead to severe clinical outcomes for both mother and child. Even if protection against the disease can gradually be acquired following successive pregnancies, the malaria parasite has developed a large panel of evasion mechanisms to escape from host defense mechanisms and manipulate the immune system to its advantage. Infected erythrocytes isolated from placentas of women suffering from placental malaria present a unique phenotype and express the pregnancy-specific variant VAR2CSA of the Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) family at their surface. The polymorphic VAR2CSA protein is able to mediate the interaction of infected erythrocytes with a variety of host cells including placental syncytiotrophoblasts and leukocytes but also with components of the immune system such as non-specific IgM. This review summarizes the described VAR2CSA-mediated host defense evasion mechanisms employed by the parasite during placental malaria to ensure its survival and persistence.
... In the present study, a specific binding protein (rVAR2) was also recombined for pl-CSA detection. The signal of rVAR2, labeled with a 6×His tag, indicating specific binding with pl-CSA, was amplified through a DyLight® 650-labeled anti-6×His antibody that showed a high specificity, consistent with previous studies [33]. This may provide an intervention or detection reagents for choriocarcinoma. ...
Background: Placental-like chondroitin sulfate A (pl-CSA) is exclusively expressed in cancerous and placental tissues and is highly correlated with the degree of malignancy. However, the mechanism through which pl-CSA regulates tumorigenesis and metastasis in choriocarcinoma remains unclear.
Methods: Stable transfectants of the JEG3 choriocarcinoma cell line, including a negative control (NC) line and a cell line with knockout of the biosynthetic enzyme CS synthase-2 (ChSy-2) (ChSy-2-/-), were obtained using CRISPR/Cas9 systems and identified by immunofluorescence, flow cytometry, western blots and enzyme-linked immunosorbent assays (ELISAs). The proliferation, migration, invasion and colony formation of the cells were determined by a cell counting kit, scratch-wound assays, transwell assays and soft agar colony formation assays in vitro, respectively. The tumorigenesis and metastasis of choriocarcinoma were also investigated through two xenograft models in vivo.
Results: The ChSy-2 protein in the ChSy-2-/-group was below the detection threshold, which was accompanied a significant reduction in the pl-CSA level. Reducing pl-CSA through ChSy-2 knockout significantly inhibited cell proliferation, migration, invasion and colony formation in vitro and tumorigenesis and metastasis of choriocarcinoma, with deceases in tumor volume and metastatic foci and a high percent survival compared to the NC in vivo.
Conclusion: pl-CSA, as a necessary component of JEG-3 cells, was efficiently reduced through ChSy-2 knockout, which significantly inhibited the tumorigenesis and metastasis of choriocarcinoma. ChSy-2/pl-CSA could be alternative targets for tumor therapy.
... The specific structure of this CS subtype, also called oncofetal CS (ofCS), remains to be fully characterized. Current evidence suggests the presence of discrete structural requirements for binding including, but not limited to, extensive 4-O sulfation Alkhalil et al. 2000;Valiyaveettil et al. 2001;Achur et al. 2008;Sugiura et al. 2016a). While some studies have suggested what PGs may carry ofCS (Ayres Clausen et al. 2016), the full repertoire of core proteins and their specific protein attachment sites remains unknown. ...
Chondroitin sulfate (CS) is the placental receptor for the VAR2CSA malaria protein, expressed at the surface of infected erythrocytes during Plasmodium falciparum infection. Infected cells adhere to syncytiotrophoblasts or get trapped within the intervillous space by binding to a determinant in a 4-O-sulfated CS chains. However, the exact structure of these glycan sequences remains unclear. VAR2CSA-reactive CS is also expressed by tumor cells, making it an attractive target for cancer diagnosis and therapeutics. The identities of the proteoglycans carrying these modifications in placental and cancer tissues remains poorly characterized. This information is clinically relevant since presentation of the glycan chains may be mediated by novel core proteins or by a limited subset of established proteoglycans. To address this question, VAR2CSA-binding proteoglycans were affinity-purified from human placenta, tumor tissue, and cancer cells, and analyzed through a specialized glycoproteomics workflow. We show that VAR2CSA-reactive CS chains associate with a heterogenous group of proteoglycans, including novel core proteins. Additionally, this work demonstrates how affinity purification in combination with glycoproteomics analysis can facilitate the characterization of CSPGs with distinct CS epitopes. A similar workflow can be applied to investigate the interaction of CSPGs with other CS binding lectins as well.
... In vertebrates, the chondroitin polysaccharide undergoes extensive modifications of sulfotransferases and chondroitin-specific epimerases (Mizumoto et al. 2013;Ly et al. 2011). This results in complex yet defined CS/DS structures that may interact with various protein ligands with different degree of specificities (Le Jan et al. 2012;Mizumoto et al. 2015;Sugiura et al. 2016). In contrast, chondroitin in C.elegans is considerably less complex and the general view was, until recently, that the nematode only produces non-sulfated chondroitin (Yamada et al. 1999;Toyoda et al. 2000). ...
Proteoglycans regulate important cellular pathways in essentially all metazoan organisms. While considerable effort has been devoted to study structural and functional aspects of proteoglycans in vertebrates, the knowledge of the core proteins and proteoglycan-related functions in invertebrates is relatively scarce, even for C.elegans. This nematode produces a large amount of non-sulfated chondroitin in addition to small amount of low-sulfated chondroitin chains (Chn and CS chains, respectively). Until recently, 9 chondroitin core proteins (CPGs) had been identified in C.elegans, none of which showed any homology to vertebrate counterparts or to other invertebrate core proteins. By using a glycoproteomic approach, we recently characterized the chondroitin glycoproteome of C.elegans, resulting in the identification of 15 novel CPG core proteins in addition to the 9 previously established. Three of the novel core proteins displayed homology to human proteins, indicating that CPG and CSPG core proteins may be more conserved throughout evolution than previously perceived. Bioinformatic analysis of the primary amino acid sequences revealed that the core proteins contained a broad range of functional domains, indicating that specialization of proteoglycan-mediated functions may have evolved early in metazoan evolution. This review specifically discusses our recent data in relation to previous knowledge of core proteins and GAG-attachment sites in Chn and CS proteoglycans of C.elegans and humans, and point out both converging and diverging aspects of proteoglycan evolution.
