Cerebral Malaria (CM) is a severe complication of Plasmodium falciparum infection, leading to significant neurological damage and remains a major public health concern despite effective antimalarial therapies. Glycophorins are essential components of the red blood cell membrane that contribute to its structure, flexibility, and interactions with pathogens. Glycophorin A (GPA) is crucial for Plasmodium falciparum invasion during malaria, making it a key target for research aimed at preventing malaria. The other glycophorins, GPB, GPC, and GPD, also play important roles in RBC membrane integrity, blood group determination, and possibly pathogen interactions. They are also involved in the sequestration of infected RBCs in the brain during cerebral malaria. By promoting cytoadherence and microvascular obstruction, glycophorins contribute to the neurological complications of CM. Research into the role of glycophorins in malaria pathogenesis may offer new therapeutic targets for preventing or treating cerebral malaria. Understanding the functions of glycophorins is vital for advancing therapies for malaria, improving blood transfusion compatibility, and enhancing our knowledge of RBC biology.

• Red Blood Cells (RBCs)
• Glycophorins
• Plasmodium falciparum
• Cerebral Malaria (CM)

Joshi U, George LB, Highland H (2024) Glycophorins and Cerebral Malaria. Ann Clin Pathol 11(2): 1174.

BBB: Blood-Brain Barrier; CM: Cerebral Malaria; CD: Cluster Domain; EBA: Erythrocyte Binding Antigen; GPA: GlycoPhorin A; GPB: GlycoPhorin B; GPC: GlycoPhorin C; GPE: GlycoPhorin E; iRBCs: infected RBCs; P. falciparum: Plasmodium falciparum; PfEMP1: Plasmodium falciparum erythrocyte membrane protein 1; RBCs: Red Blood Cells

According to World Malaria report, in 2022, there were an estimated 247 million malaria cases worldwide, and around 619,000 deaths due to malaria. The majority of malaria cases and deaths continue to be concentrated in sub-Saharan Africa, which accounts for about 95% of all cases and 96% of deaths [1]. Although the number of malaria cases worldwide has stayed mostly constant since 2020, a number of variables have raised the danger of malaria incidence, including pesticide resistance, climate change, the emergence of treatment resistance to artemisinin, and many more [2]. There are five primary Plasmodium species that infect humans and cause malaria, including Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi [3,4]. Each species has different characteristics in terms of disease severity, geographic distribution, and transmission dynamics, but they all rely on the Anopheles mosquito as their vector for transmission [3]. Among these five, Plasmodium falciparum is the most common and dangerous species, responsible for most severe malaria cases and deaths worldwide. It is prevalent in sub-Saharan Africa, Southeast Asia, and parts of the Pacific [5].

Glycophorins are sialoglycoproteins, a family member of transmembrane proteins that are primarily found in the membrane of Red Blood Cells (RBCs). They are integral membrane proteins that play key roles in the structural integrity of the RBC membrane, in maintaining the cell’s mechanical properties, and in mediating interactions with other molecules, including pathogens [6]. Glycophorins are important not only for the general functions of RBCs but also as receptors for certain diseases, particularly malaria [7]. There are four main glycophorins found in the RBC membrane: Glycophorin A (GPA), Glycophorin B (GPB), Glycophorin C (GPC), and Glycophorin D (GPD). Each of these glycophorins has a distinct structure, function, and role in RBC physiolog [8]. General Functions of Glycophorins in RBCs include 1) The glycophorins, particularly GPA, help anchor the RBC’s cytoskeleton to the plasma membrane. This anchoring is critical for maintaining the biconcave shape and deformability of RBCs, which allows them to traverse the narrowest capillaries in the circulatory system. 2) Glycophorins contribute to the negative charge on the RBC surface through the glycosylation of their extracellular domains. This repulsive charge helps prevent RBCs from clumping together (aggregation), promoting smooth blood flow [9]. 3) MNS blood group system is one of the major human blood group systems and is defined by the presence of specific antigens/glycophorins on the surface of red blood cells (RBCs). Blood Group Antigens are important in blood transfusion compatibility and in determining individual differences in immune response [10]. 4) Glycophorins serve as receptors for pathogens like Plasmodium falciparum (P. falciparum) and the Duffy antigen (which is used by some strains of malaria parasites for invasion). These interactions are crucial for the parasitic life  cycle and have implications for the development of therapies aimed at blocking these pathways [11].

Structure and functions of Glycophorins

Glycophorins A (GPA): The most prevalent glycophorin A (GPA) is found in 0.5 million copies per red blood cell. It has a single transmembrane region, a short cytoplasmic tail, and a sizable extracellular domain full of carbohydrate chains containing sialic acid [12].

GPA contributes to the overall stability and flexibility of the RBC membrane. The highly glycosylated extracellular portion helps the RBC maintain a negative surface charge, which is important for preventing RBC aggregation and for promoting blood flow. GPA is a major receptor for the P. falciparum ligand EBA-175 ligand [13]. The M antigen and the N antigen are two alleles of the same gene and are differentiated by a single amino acid substitution in the extracellular domain of GPA. Where M and N antigen are characterized by serine and asparagine at position 1 of the extracellular domain of GPA respectively. Hence, The MNS blood group system is based on the polymorphisms in GPA (M and N antigens), which result from variations in the extracellular part of the protein [14]. Through its eight transmembrane helices, Band 3 is linked to GPA, creating a dual protein that results in the so-called Wrb blood group antigen [15]. Based on the previous studies MSP-1-band 3/GPA complex considered as a key component in the development of the during the first adhesion phase of P. falciparum’s RBC invasion. This interaction is crucial for the parasite’s lifecycle, making GPA a significant target in malaria research [16].

Glycophorin B (GPB): GPB is a less abundant glycoprotein compared to GPA but is still an important part of the RBC membrane. It shares similar structural features with GPA, including a transmembrane domain and extracellular carbohydrate chains [17]. GPB is the basis of the MNS blood group system with the S and s antigens, which are distinct from those defined by GPA. Whereas the S antigen and the s antigen are variants of the GPB gene and are determined by the presence of specific amino acid substitutions at position 29 of the GPB protein. Where S antigen is characterized by glycine at position 29 while s antigen is characterized by serine at position 29 [18]. However less involved in malaria compared to GPA, GPB may also play a minor role in the cytoadherence of infected RBCs, though its function in pathogen interactions is less studied than GPA. P. falciparum EBL-1 ligand binds to GPB, as well glycophorin B-PfEMP1 interaction mediates rosetting in P. falciparum infection [19].

Glycophorin C (GPC): Glycophorin C (GPC) is a single-pass transmembrane protein with a large extracellular domain, a short transmembrane segment, and a small cytoplasmic tail. It is much smaller than GPA and GPB. The gene GYPC encodes both glycophorin C and glycophorin D. not related to GYPA or GYPB. GPC plays a role in the structural integrity of the RBCs but is not known to serve as a major receptor for pathogens like GPA. GPC acts as a receptor for P. falciparum EBA-140 ligand, with successful binding reliant on sialic acid residues in N- and O-linked oligosaccharides of GPC [6]. GPC is also involved in anchoring the RBC cytoskeleton (specifically, spectrin and actin) to the membrane via interactions with protein 4.1. This helps maintain RBC shape and deformability, which are essential for RBC function in the circulatory system. GPC carries the Gerbich blood group antigens, which are less clinically relevant but still important in blood transfusion compatibility [20].

Glycophorin D (GPD): Glycophorin D (GPD) is a more recently discovered glycophorin, structurally similar to GPC but with a slightly different extracellular domain. GPD contributes to the stability of the RBC membrane and is involved in the overall organization of the glycophorin family in the membrane. It is suggested that GPC is a novel P. falciparum receptor in humans, whereas GPD is an ancestral EBA-140 receptor in non-human primates [21]. GPD is associated with the Dombrock blood group system and is important in transfusion medicine, even though its involvement in pathogen interactions is less clear than those of GPA or GPB [9].

Since no protein product for glycophorin E has been found, the gene designated as GYPE, which is extremely similar to GYPA and GYPB, is probably a pseudogene [9]. Some previous studies reported that complete lacking GPA and GPB proteins confer protection against falciparum malaria [22,23]. Variations in the glycophorin family contribute to different blood group systems, such as MNS, Dombrock, and Gerbich, which are important for transfusion medicine. Blood group antigens on glycophorins are key determinants of blood compatibility and are important for immune reactions in transfusion settings [24]. The most notable role of glycophorins in disease is in malaria because they serve as a primary receptor for the malaria parasite. The interaction between Plasmodium and glycophorins is critical for the invasion of the RBC by the Plasmodium spp [9]. Non allelic homologous recombination between the glycophorin genes (GYPA, GYPB and GYPC) resulted into one allele called DUP4. DUP4 found as a structural genomic variant that carries extra copies of a glycophorin A-glycophorin B fusion gene. Which confers protection against malaria by reducing the risk of severe malaria up to 74% in homozygous individuals. Additionally in falciparum malaria endemic regions DUP4 has found to be associated with elevated Haemoglobin levels and RBC surface tension [7,12].

Glycophorins and Cerebral Malaria

Cerebral malaria (CM) is the most severe complication of falciparum, malaria, often fatal for children, pregnant women and non-immune travellers. Apart from P. falciparum, P. vivax infections can also result in CM [25] while P. knowlesi can sometimes induce severe symptoms of malaria [26]. In CM, iRBCs bind to endothelial cells in blood vessels, especially in the small vessels of the brain. This process is known as cytoadherence. The cytoadherence of iRBCs to the blood-brain barrier (BBB) plays a critical role in the development of cerebral malaria. The iRBCs can adhere to the endothelial cells lining the brain’s microvasculature, obstructing blood flow and leading to inflammation, seizures, coma, hypoxia, and tissue damage in the brain, and death if untreated [27,28]. The pathogenesis of CM is complex. It includes parasite sequestration where iRBCs adhere to the brain’s microvascular endothelial cells, a process enhanced by the expression of certain receptors on both iRBCs and the endothelium. This sequestration of infected RBCs leads to microvascular obstruction and impaired blood flow, contributing to hypoxia and cerebral edema [29]. Additionally, infection with P. falciparum induces endothelial cell dysfunction, leading to blood-brain barrier (BBB) disruption, inflammation, and neuroinflammation. This results in cognitive deficits and other neurological impairments. Inflammatory response, in which the accumulation of iRBCs in the brain triggers local inflammation. This leads to the activation of the immune system, which produces pro-inflammatory cytokines (e.g., TNF-α, IL-1), contributing to BBB disruption and worsening the conditions. The interaction between glycophorins and Plasmodium invasion and sequestration contributes to the inflammation and endothelial cell damage seen in CM [30,31]. 3) The cytoadherence of iRBCs to brain vasculature and the associated immune responses can cause oxidative stress, leading to tissue damage. The resultant brain injury may lead to neurological complications, including seizures and coma [32]. Glycophorins, particularly Glycophorin A, are involved in the interaction between iRBCs and endothelial cells. These interactions are mediated through a variety of adhesion molecules and ligands expressed on both the RBCs and the endothelial cells. PfEMP1 on the surface of iRBCs can bind to several receptors on endothelial cells, including CD36, ICAM-1, and endothelial protein C receptor (EPCR) [33,34]. These receptors, which are upregulated during Plasmodium infection, interact with glycophorin family members, aiding in the sequestration of iRBCs in the brain. The sequestration of iRBCs in the brain contributes to the pathology of cerebral malaria by obstructing blood flow, causing localized inflammation, and leading to neuronal damage and cerebral edema [29]. Researchers are investigating glycophorin-mediated treatment approaches that target the connection between Plasmodium and glycophorins and act on the metabolism of carbohydrates, given the pivotal role that glycophorins play in the invasion and pathogenesis of malaria [35]. Glycophorin A-deficient RBCs demonstrated verified resistance to malaria, but glycophorin B-deficient RBCs shown partial resistance. This demonstrates the importance of glycophorin A. Additionally, variation in both glycophorin A and B demonstrated resistant proved role of both the glycophorins in Plasmodium invasion [36]. Antibodies against Glycophorin A could inhibit the Plasmodium invasion [36], vice a versa antibodies against region II of EBA-175 (ligand of Glycophorin A) could reduce the Plasmodium invasion into erythrocytes by 50% [37]. Autoantibodies were also reported in falciparum malaria endemic regions reported by [38]. Antibodies against the BRIC 4 region of CD236R significantly inhibited rosette formation, supporting the role of glycophorin C in P. vivax rosetting. While understanding the role of glycophorin C in rosetting can help in the search for the P. vivax rosette ligand and provide insights into malaria pathogenesis and potential therapeutic targets [39]. Hence, blocking of glycophorin interactions can be a potential strategy, where we develop antibodies or small molecules that block the binding of Plasmodium falciparum to glycophorin A, thereby preventing invasion of RBCs although further studies are required [31]. Future aspects include genetic approaches to alter glycophorin expression or function might be another strategy, though this would involve significant challenges related to safety and efficacy. Also, development of vaccine which targets the Plasmodium proteins that interact with glycophorins could also reduce the incidence of malaria and its severe forms, including cerebral malaria.

In summary, glycophorins, particularly glycophorin A and B play a critical role in malaria. They are crucial for the invasion of Plasmodium into RBCs, and they are also involved in the sequestration of infected RBCs in the brain during cerebral malaria. By promoting cytoadherence and microvascular obstruction, glycophorins contribute to the neurological complications of CM. Research into the role of glycophorins in malaria pathogenesis may offer new therapeutic targets for preventing or treating cerebral malaria.

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