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Tumor and Cell Biology (MTC), Karolinska Institutet, Box 280, Nobels väg 16, SE-171 77. 8 ... using synthetic ABO-decorated giant unilamellar ves...
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ABO Blood Group Antigen-Decorated Giant Unilamellar Vesicles Exhibit Distinct Interactions with Plasmodium falciparum-Infected Red Blood Cells Charikleia-Despoina Vagianou, Nicolai Stuhr-Hansen, Kirsten Moll, Nicolai Bovin, Mats Wahlgren, and Ola Blixt ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00635 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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ABO Blood Group Antigen-Decorated Giant Unilamellar Vesicles Exhibit Distinct Interactions with Plasmodium falciparum-Infected Red Blood Cells

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Charikleia-Despoina Vagianou†, Nicolai Stuhr-Hansen†, Kirsten Moll‡, Nicolai Bovin§, Mats Wahlgren‡, Ola Blixt†*

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† Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. E-mail: [email protected] ‡ Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Box 280, Nobels väg 16, SE-171 77 Stockholm, Sweden § Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation Supporting Information Placeholder

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ABSTRACT: Severe malaria is considered to be the deadliest disease of the current century, primarily among children in sub-Saharan Africa. It stems from infection by the virulent parasite Plasmodium falciparum. The pathogenesis of the disease is based on the rosetting phenomenon, which occurs during the life cycle of the parasite in red blood cells (RBCs) and promotes the binding of parasitized RBCs to healthy ones. The role of the ABO blood group antigens in relation to the phenomenon has previously only been investigated in clinical isolates obtained from malaria patients. Here, we aim to clarify their role using synthetic ABO-decorated giant unilamellar vesicles (GUVs), which serve as simple biomimetic models of RBC-size cell membranes. Our results suggest clearly and for the first time that the blood group A and O antigens have a direct impact on receptor-specific rosetting phenomena when compared to the B antigen, which only participates in rosetting to an insignificant degree. Thus, glycodecorated GUVs represent a practical tool for studying cell-surface interactions.

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Plasmodium falciparum, which is the most virulent species of the genus Plasmodium, causes a lethal form of malaria. The parasite is transmitted to the human host via a mosquito vector, and once it invades the body, it develops in different stages, infecting the liver as a sporozoite before multiplying and releasing thousands of merozoites into the bloodstream in order to further infect red blood cells (RBCs).1 When P. falciparum invades RBCs, it promotes the binding between P. falciparum-parasitized RBCs (pRBCs) and uninfected ones, thereby leading to a cell aggregation phenomenon known as rosetting.2 This phenomenon is an important adhesion phenotype, and it is associated with the virulence of the parasite, as well as the severity of malaria cases3–5, due to obstructing blood flow in the microvasculatory system.6 Rosetting and

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sequestration have been suggested to help the parasite evade recognition by the host’s immune system. Indeed, sequestration to the endothelial cells of the blood vessels protects against clearance by the spleen, while rosetting, whereby an pRBC surrounds itself with uninfected RBCs, renders the parasite-derived antigens inaccessible to antibodies, which in turn leaves the immune system unable to fight the parasite.7 Both mechanisms are mediated by three major variant surface antigen (VSA) families that the parasite expresses on the parasitized RBC membrane: P. falciparum erythrocyte membrane protein 1 (PfEMP1),8 repetitive interspersed repeats (RIFINs),9 and subtelomeric open reading frame (STEVOR).10 Over the past few years, extensive studies involving clinical isolates conducted in different parts of the world have correlated the severity of malaria seen in different countries with the ABO blood group of the patient. More specifically, the invasion of the parasite differs between patients with different blood groups (ABO), with blood group A individuals having a greater probability of developing the severe form of the disease.11–14 On the erythrocyte surface, the A and B antigens are terminal trisaccharides A, GalNAcα(1,3)[(Fucα1→2)]Galβ1-, and B, Galα(1,3)[(Fucα1,2)]Galβ1-, respectively, which are attached to glycoproteins and glycolipids. Blood group O individuals lack the glycosyltransferases necessary to produce the A or B antigens, and they carry the H antigen, namely the disaccharide Fucα1→2Galβ1.15 The formation of rosettes involves ABO blood group-independent receptors, such as complementary receptor 1 (CR1)16 or heparan sulfate.17 In addition, the rosettes vary in size depending on the preferences of the ABO antigens. Several studies have shown that larger rosettes with the ability to better withstand disruption are formed between pRBCs and the RBCs of blood group A, B, or AB, while blood group O RBCs are significantly smaller.18–20 This indicates the potential involvement of A and B antigens in the rosetting phenomenon due to acting as the binding sites of RBCs for adherence to the VSA of the

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Scheme 1. BODIPY-labelled cholesterylated glycopeptide structures incorporated into the giant unilamellar vesicle (GUV) membrane. pRBCs. Further, recent studies have suggested that RIFINs play an important role in ABO glycan-dependent rosetting via the glycophorin A receptor.9 The role of STEVOR in the rosetting phenomenon is the least studied aspect in this regard, although Niang et al. reported that binding between STEVOR and glycophorin C leads to rosetting in a PfEMP1-independent manner.10 In order to gain a better understanding of the interactions of the ABO blood group antigens with pRBCs, we aimed to build a simple synthetic RBC-size membrane containing selected ABO glycan used onto giant unilamellar vesicles (GUVs). GUVs have been widely used in multiple studies not only as membrane models, but also because their size being comparable to cell size, their flexibility, and their finite volume render them suitable for use as cell models.21 Moreover, unlike RBCs, GUVs offer the unique opportunity to generate a vesicle decorated with selected glycan ligands on the surface, thereby allowing us to characterize the specific interaction between a pRBC and the parasite receptor. The previously introduced amphipathic function-spacer-lipid (FSL) Kode constructs that carry blood group antigens have been used for RBC decoration.22 However, the use of a chemical module composed of fluorescently labeled cholesterylated peptide modules with alkyne functionalities allowed us to introduce any azido-glycan of choice and, additionally, provided the opportunity to monitor their behavior in artificial membranes. In the present study, we investigate the interactions between different ABO-GUVs and pRBCs of P. falciparum (FCR3S1.2 strain), as well as the interactions after specific antibody blocking against PfEMP1 and the A-RIFIN RBC receptor. Results and Discussion Rosette formation has been proven to be dependent on specific human serum components that act as bridging molecules between healthy and parasitized RBCs.23 Among these components, IgM, the natural antibody in the first line of defense against invading pathogens, plays a crucial role due to mediating the binding of the PfEMP1 of pRBCs with host cell receptors on uninfected RBCs.24,25 Thus, for in vitro experiments, the addition of serum is of great importance in relation to the observation of rosetting (Fig. S2a). However, the addition of serum to our GUV modules induced their aggregation (Fig. S2b). This limitation led us to add intravenous (IV) Igs, which contain IgM (Fig. S1), rather than serum in order to mediate the IgM-dependent binding. The IV Igs (1 mg) were diluted in sucrose aqueous solution (290 mM). Further dilution (1:10) then induced sufficient rosetting in the pRBCs (Fig. S2c), while the behavior of the GUVs was not affected (Fig. S2d). Thus, IV Igs can be used to efficiently induce rosetting without the presence of serum, which allows us to observe the interactions of glycodecorated GUVs with the rosettes of pRBCs without complications. All the BODIPY-labelled cholesterylated glycopeptides utilized in this study were prepared by means of tandem click chemistry according to our recently described procedure.26 A bicyclononyne (BCN)-BODIPY fluorophore unit was attached

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to a cholesterylated azido-alkyne peptide using a strainpromoted alkyne-azide cycloaddition (SPAAC) and then further functionalized with a range of azido-glycans27 using a copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). Six different BODIPY-labelled cholesterylated glycopeptide modules (Scheme 1) were inserted into the GUV membrane in order to investigate the interactions, first with glycan-specific lectins and then with pRBCs. Mixtures of 1,2-Dioleoyl-snglycero-3-phosphocholine (DOPC), cholesterol, the membrane marker Lissamine™ Rhodamine B 1,2-Dihexadecanoylsn-Glycero-3-Phosphoethanolamine (RhoB-DHPE), and BODIPY-labelled cholesterylated glycopeptides were used for the GUV production.

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Figure 2. Interactions of the parasitized RBCs (pRBCs) with the glycodecorated GUVs after the addition of IV Igs to induce rosetting. Mixtures containing DOPC/cholesterol were used for the GUV electroformation protocol involving (a) 5 mol% blood group A, 3’SLN, 6’SLNLN, or GN-BODIPYlabelled cholesterylated peptide (green) , 60 x magnification

Figure 1. Fluorescent images of the ABO-decorated GUVs and the ABO antigen presence on the GUV surface verified by RSL/streptavidin recognition. The ABO-GUVs were produced by a mixture of DOPC/cholesterol (90:5) and 5 mol% BODIPY-FL-labelled (green) a) blood group A, b) blood group B, and c) blood group O cholesterylated peptide. All three antigens present on the ABO-GUVs were accessible for the biotinylated Ralstonia solanacearum lectin (RSL) and streptavidin (d-f). Scales at 10 µm. All the modules were incorporated into the GUVs, as indicated by the BODIPY FL marker showing an equal visual distribution around the lipid membrane. The viability and accessibility of the different glycans, namely GlcNAc (GN), Neu5Acα(2,3)Galβ(1,4)GlcNAc (3’SLN), and NeuAc5Acα(2,6)Galβ(1,4)GlcNAcβ(1,3)Galβ(1,4)GlcNAc(6’SLNLN), on the GUV surface has previously been demonstrated, assembled, displayed, and recognized by glycanrecognizing corresponding lectins.26 The three incorporated cholesterylated ABO modules were detected using biotinylated fucose-binding Ralstonia solanacearum lectin (RSL)28 and streptavidin-AF555. Each ABO-GUV was agglutinated by the RSL-Streptavidin complex, thereby verifying the ABO modules’ incorporation into the GUV membrane (Fig. 1). All three ABO-GUVs, as well as a negative control (module without glycan), were next incubated with uninfected A, B, or O RBCs for 5, 10, and 30 minutes at room temperature in the presence or absence of IV Igs. None of the ABO-GUVs interacted with the non-infected RBC cell membrane regardless of the blood group antigen presence (Fig. S3). Instead, the blood group O RBCs infected with the laboratory parasite clone FCR3S1.2 of a rosetting phenotype (pRBC) specifically interacted with the A-GUVs, but not with the GUVs decorated with the non-blood group glycans 3’SLN, 6’SLNLN, and GN (Fig. 2a). Interestingly, the blood group O-GUVs did participate in the rosetting, although the B-GUVs clearly did not, which indicates that the blood group B antigen played no primary role in the formation of rosetting in this in vitro assay (Fig. 2b).

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lens, interactions with formatted rosettes of pRBCs-RBCs; and (b) 5 mol% membrane marker RhoB-DHPE (red), blood group A, B, or O BODIPY-labelled cholesterylated peptide (green), 100 x magnification lens, interactions with formatted rosettes of pRBCs (blue, parasitic DNA staining)-RBCs.

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suggested. Furthermore, we propose that the PfEMP1 ligand also interacts with the O antigen, which has not been demonstrated previously. A visual quantification of the number of rosettes in which the A-, B-, or O-GUVs participated revealed blood group A to have significant involvement (Fig. 3c).

The PfEMP1 protein, which is encoded by the var gene family, is the best characterized of the VSA families, and it is known to bind to a variety of host cell receptors primarily found on endothelial vascular cells. Each parasite genome contains around 60 different var genes, of which only one is expressed at a time,

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thereby mediating binding with one or more different host receptors concurrently. CD36, intercellular adhesion molecule 1 (ICAM-1),29 chondroitin sulfate A (CSA),30 and heparin-like carbohydrates17 have all been identified as receptors of PfEMP1 leading to the occurrence of ABO blood group independent sequestration, while rosetting is mediated by the CR1 and heparan sulfate present on the RBC surface. Although prior studies have shown that the blocking of PfEMP1 efficiently disrupts the rosettes of blood group O pRBCs, the role of ABO antigens in rosetting has not yet been fully determined.

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Finally, we investigated whether our glycomodules could be embedded onto the surface of infected blood group O pRBCs carrying either A or B antigens. Both were efficiently incorporated into the pRBCs membrane, as indicated by the BODIPY FL marker (Fig. 3b). The antibody blocking of the PfEMP1 and the subsequent spiking of the pRBCs with the B-antigen module revealed that the alteration of the membrane by this antigen did not promote any interactions between cells. However, the inserted blood group A-antigen module forming an A-type O-pRBC promoted the formation of rosettes. As time elapsed, the formation of rosettes increased in terms of both number and size (Fig. 3b).

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The efficient disruption of rosetting in the blood group O pRBCs (Fig. S2b) was accomplished using the polyclonal purified IgG fraction against PfEMP1 (ITvar60), and two distinct experiments were performed in order to elucidate the role of the A antigen in rosetting. First, upon IV Igs-induced rosetting, our O-GUVs showed no interaction with the pRBCs in the presence of anti-PfEMP1. The B-GUVs’ behavior in the rosettes did not change following the addition of antiPfEMP1, since they appear to not interact with the formatted rosettes. Yet, the A-GUVs were found to still bind to the pRBCs following the addition of the antibody (Fig. 3a)

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In summary, we prepared cholesterylated peptide modules functionalized with ABO blood group glycans displayed on GUVs in order to determine their participation in pRBC rosetting. By using ligand-specific polyclonal antibodies, we demonstrated that blood groups O and A play a primary role in the formation of rosettes, while blood group B has no primary role. More specifically, the

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RIFIN-A of the parasite clone FCR3S1.2 acts as a mediator for blood group A and PfEMP1 takes part in the rosetting of O antigens, but not for blood group B. Glycodecorated GUVs carrying a variety of different glycosylated peptides have the potential to become the method of choice for studying the role of carbohydrates in cell-cell or cell-pathogen communication. Additionally, cholesterylated glycopeptides represent a new horizon for carbohydrate-targeted drug delivery through the engineering of biological membranes.

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blood group A rosetting. As RIFINs are also expressed on the pRBC surface of the parasite clone FCR3S1.2 and involved in rosetting,9 we included anti-RIFIN-A antibodies to disrupt the rosettes (Fig. S2c). First, the anti-RIFIN-A antibody alone did not disrupt the rosetting of the GUVs containing blood group A or blood group O modules, while the B-GUVs did not exhibit any involvement in the rosettes, which was as expected (Fig. 3a). However, when both receptors, namely PfEMP1 and RIFIN-A, were blocked by antibodies, none of the three antigen-decorated GUVs (A, B, and O) participated in the rosetting (Fig. 3a). Our findings suggest that RIFIN-A is not the only ligand on the pRBCs to interact with the blood group A antigen and thereby lead to blood group A-dependent rosetting; however, PfEMP1 binding to this antigen is also

Therefore, these observations verify previous findings sug290 ASSOCIATED CONTENT gesting that PfEMP1 is not the only protein responsible for rosetting and, further, that PfEMP1 is not directly involved in Figure 3. (a) Interactions of blood group O pRBCs with the ABO-GUVs after the addition of the PfEMP1 antibody, RIFIN-A antibody, or both in the presence of IV Ig. Mixtures containing DOPC/cholesterol were used for the GUV electroformation protocol involving RhoBDHPE (membrane marker), 4.9 mol% blood group O cholesterylated peptide, 4.9 mol% blood group A cholesterylated peptide, and 4.9 mol% blood group B cholesterylated peptide (green, BODIPY FL). DAPI (blue) stained the parasite’s nuclei. Scale bars, 10 µm. Arrows indicate A-, B-, or O-GUVs participating in rosettes. (b) Spiking of the pRBCs, after the anti-PfEMP1 treatment, with blood group A or blood group B cholesterylated peptide (green, BODIPY FL). Scale bars, 10 µm. (c) % participation of the ABO-GUVs in rosetting without treatment and upon treatment with anti-PfEMP1, anti-RIFIN-A, or both. The results are shown as the mean of the independent measurements ± SE (n = 3). Comparison between the ABO-GUVs in terms of binding to the pRBC rosettes, as analyzed using a one-way analysis of variance (ANOVA); *P < 0.05, **P < 0.01.

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Supporting Information Supporting information is available containing experimental details and results. This material is available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION

Corresponding Author Professor Ola Blixt, PhD, University of Copenhagen Department of Chemistry, T422, Thorvaldsensvej 40, 1871, Frederiksberg, Copenhagen, Denmark. E-mail: [email protected]. Phone: +46761486838

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Notes The authors declare no competing financial interests.

305 306 ACKNOWLEDGMENT 307 The authors wish to acknowledge the support provided by the 308 Danish Research Council (Innovationsfonden) and the frame309 work of the EU ERASynBio project SynGlycTis, as well as 310 the support offered by the Stiftelsen for Strategisk Forskning 311 (SSF). The Consortium for Functional Glycomics (CFG) is 312 also to be thanked for providing the azido glycans, as is Prof. 313 Brigit Wiltrich (Austria) for providing the biotinylated RSL. 314 REFERENCES 315 316 (1) Bousema, T., Okell, L., Felger, I., and Drakeley, C. (2014) 317 Asymptomatic malaria infections: Detectability, transmissibil318 ity and public health relevance. Nat. Rev. Microbiol. 12, 833– 319 840. 320 321 322

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(30) Rogerson, S. J., Chaiyaroj, S. C., Ng, K., Reeder, J. C., and Brown, G. V. (1995) Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes. Exp. Med. 182, 15–20.

TOC: Schematic representation of the decoration of GUVs with fluorescently labelled cholesterylated glycopeptides and their involvement in rosetting through binding to Plasmodium-infected RBCs.

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Figure 1 208x211mm (96 x 96 DPI)

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Figure 2 68x48mm (220 x 220 DPI)

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Figure 3 295x164mm (96 x 96 DPI)

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Scheme 1 164x102mm (150 x 150 DPI)

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TOC 290x148mm (96 x 96 DPI)

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