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Structural variants are mapped that are correlated with a reduced risk of severe malaria. Large-scale deletions and duplications of genes, referred to as structural variants (SVs), are common within the human genome and have been linked to disease. Examining a genomic region that appears to confer a selective benefit, Leffler et al. used fine mapping to identify a specific SV that reduces the risk of severe malaria by an estimated 40% (see the Perspective by Winzeler). Data from African individuals revealed that populations harbor different SVs in this region. Furthermore, by dissecting a highly complex genomic region, the authors identified the likely causal element. This element encodes hybrid genes that affect glycophorin proteins, which are used by the malarial parasite in infection and are associated with resistance to severe disease. Science , this issue p. eaam6393 ; see also p. 1122 The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB . We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

Glycophorins are transmembrane proteins of red blood cells (RBCs), heavily glycosylated on their external-facing surface. In humans, there are four glycophorin proteins, glycophorins A, B, C and D. Glycophorins A and B are encoded by two similar genes GYPA and GYPB, and glycophorin C and glycophorin D are encoded by a single gene, GYPC. The exact function of glycophorins remains unclear. However, given their abundance on the surface of RBCs, it is likely that they serve as a substrate for glycosylation, giving the RBC a negatively charged, complex glycan “coat”. GYPB and GYPE (a closely related pseudogene) were generated from GYPA by two duplication events involving a 120-kb genomic segment between 10 and 15 million years ago. Non-allelic homologous recombination between these 120-kb repeats generates a variety of duplication alleles and deletion alleles, which have been systematically catalogued from genomic sequence data. One allele, called DUP4, encodes the Dantu NE blood type and is strongly protective against malaria as it alters the surface tension of the RBC membrane. Glycophorins interact with other infectious pathogens, including viruses, as well as the malarial parasite Plasmodium falciparum, but the role of glycophorin variation in mediating the effects of these pathogens remains underexplored.

Serological typing of MNS polymorphic antigens - M, N, S and s - remains a fundamental technique in transfusion medicine and prenatal care, providing essential information for matching blood donors and recipients and managing haemolytic disease. Although this method is well proven and routinely used, it is not a comprehensive solution, as it has several weaknesses. Alternatively, multiplex polymerase chain reaction (PCR) is a commonly used genotyping tool due to its potency and ability to amplify several DNA targets simultaneously in a single reaction. In this work, we aimed to develop multiplex PCR and evaluate its performance for GYPA*M, GYPA*N, GYPB*S, and GYPB*s allele identification using serological and DNA sequencing methods. We also aimed to investigate the correlation between these alleles and Mi -associated hybrid glycophorins (GPs). Remarkably, multiplex PCR was well optimised, and the results aligned with serological phenotyping and DNA sequencing data with maximum accuracy and reliability; this confirmed our findings on its validity in predicting MNSs phenotypes. In addition, this work strongly demonstrates, for the first time, a moderate correlation between the GYPA*M/M and GYPB*s/s genotypes and Mi -associated hybrid GPs among Thai donors. Individuals with the GYPA*M/M and GYPB*s/s genotypes, predicted M + N - S- s + phenotypes, will thus most likely to express the Mi(a+) antigen. Nevertheless, further studies are required to validate these results and elucidate the underlying correlations.

Malaria caused by Plasmodium is responsible for approximately 250 million clinical cases and 600,000 deaths per year, mostly in Africa. Our understanding of Plasmodium parasite biology remains incomplete. The key step of Plasmodium invasion is the blood stage, which is mediated by Erythrocyte binding-like (EBL) and Reticulocyte binding-like (RBL) proteins. Three P. falciparum EBL proteins and their receptors are functional: EBA-175 – Glycophorin A (GPA), EBL-1 - Glycophorin B (GPB), and EBA-140 - Glycophorin C (GPC). Because the fourth EBA-181 ligand recognizes the mysterious erythrocyte receptor Z, which remains unknown, we aimed to characterize its specificity and search for its receptor. The Surface Plasmon Resonance method was employed to evaluate the interaction of the recombinant EBA-181 ligand with sugar moieties and the Rh2b ligand. We have demonstrated that the EBA-181 ligand binds to Neu5Ac and Neu5Gc sialic acids, interacting with the Rh2b protein with a dissociation constant of approximately 3 µM. Moreover, the EBA-181 protein binds to about 100 kDa erythrocyte membrane protein. The P. falciparum EBA-181 merozoite ligand binding was shown to be sialic acid-dependent. It seems that the Rh2b merozoite protein might be the co-ligand cooperating with EBA-181 in erythrocyte invasion. Finally, our studies suggested that erythrocyte Band 3 protein may be a putative receptor for the EBA-181 ligand.

Anion exchanger-1 (AE1) is the main erythroid Cl−/HCO3− transporter that supports CO2 transport. Glycophorin A (GPA), a component of the AE1 complexes, facilitates AE1 expression and anion transport, but Glycophorin B (GPB) does not. Here, we dissected the structural components of GPA/GPB involved in glycophorin-AE1 trafficking by comparing them with three GPB variants—GPBhead (lacking the transmembrane domain [TMD]), GPBtail (mainly the TMD), and GP.Mur (glycophorin B-A-B hybrid). GPB-derived GP.Mur bears an O-glycopeptide that encompasses the R18 epitope, which is present in GPA but not GPB. By flow cytometry, AE1 expression in the control erythrocytes increased with the GPA-R18 expression; GYP.Mur+/+ erythrocytes bearing both GP.Mur and GPA expressed more R18 epitopes and more AE1 proteins. In contrast, heterologously expressed GPBtail and GPB were predominantly localized in the Golgi apparatus of HEK-293 cells, whereas GBhead was diffuse throughout the cytosol, suggesting that glycophorin transmembrane encoded an ER/Golgi retention signal. AE1 coexpression could reduce the ER/Golgi retention of GPB, but not of GPBtail or GPBhead. Thus, there are forward-trafficking and transmembrane-driven ER/Golgi retention signals encoded in the glycophorin sequences. How the balance between these opposite trafficking signals could affect glycophorin sorting into AE1 complexes and influence erythroid anion transport remains to be explored.

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers. Cells are defined by a thin membrane that separates the inside of the cell from the outside. The core of this membrane is hydrophobic, meaning that it repels water. Many signals and nutrients cannot pass through the membrane itself, but can pass through the proteins that span the membrane. Membrane proteins are therefore essential for living cells; yet even after decades of research, it remains unclear how proteins interact with the membrane and which features determine a protein’s stability in a biological membrane. Since the early 1980s it was known that the bacterium E. coli could grow on a common antibiotic called ampicillin if it had enough of an antibiotic-degrading enzyme called β-lactamase anchored into its inner membrane. Now, Elazar et al. have used this enzyme to obtain detailed information on the interactions between a biological membrane and a membrane protein. First, hundreds of different mutations were introduced into the gene that encodes the enzyme to generate a population of bacteria that each had a slightly different membrane anchor. The mutant bacteria were then grown in the presence of the antibiotic, meaning that those mutants with a more stable membrane anchor were more likely to survive and grow than those with less stable anchors. Elazar et al. then collected all the surviving bacteria, sequenced their DNA and measured how common the different mutations were in the final population. This approach was less labor-intensive and more accurate than traditional methods for monitoring membrane-anchored proteins, and the resulting large dataset was used to uncover which features affect a protein’s stability in a membrane. These results also showed that a biological membrane’s core is considerably more hydrophobic than was previously thought. In addition to being hydrophobic, biological membranes have more negative charge in the side that faces into the cell. This means that membrane proteins with a positive charge in this region will be more stable, and Elazar et al. were able to use their new system to measure this effect for the first time. Finally, membrane proteins do not only span the membrane; they also bind with other membrane proteins in order to carry out their roles. Elazar et al. used their system to look at the surfaces of human membrane proteins that interact with one another, and build a detailed map of the interaction surfaces, from which they derived accurate models of the membrane proteins. Overall, these new findings could now be used to model the three-dimensional structures of membrane proteins and improve their stability. This in turn may help efforts to develop these proteins into more robust experimental tools and in the search for drugs that target membrane proteins.

In forensic pathology, glycophorin A (GPA) presence in tissues has been studied through anti-GPA monoclonal antibodies with immunohistochemical techniques. The use of anti-GPA in distinguishing ante- from postmortem lesions, particularly in advanced stages of decomposition, is explored in this study. This systematic review assesses the current use of glycophorins, focusing on their application in forensic investigations to detect vital hemorrhagic infiltration. A total of 799 studies were identified, and after screening, 16 studies were included, encompassing case reports, experimental studies, and case-control analyses. The data extracted from these studies highlighted the diagnostic accuracy of GPA immunohistochemical staining in forensic cases. Of the 50 specimens analyzed, 48 were stained with anti-GPA serum and 2 with anti-glycophorin C (GPC) serum. The results showed that GPA staining was significantly more reliable in detecting vital hemorrhage compared to routine histology, which had a diagnostic accuracy of only 66%. Moreover, in an experimental study, GPA positivity was observed in 72.2% of bruises and vital tissues, even in highly decomposed specimens. The study further emphasized the robustness of GPA in distinguishing ante- from postmortem injuries, with particular relevance for cases involving significant decomposition. Overall, GPA’s high sensitivity in detecting vital hemorrhage, especially in decomposed bodies, underscores its potential as a reliable tool in forensic pathology. Despite some limitations due to the small number of studies, the evidence supports the inclusion of anti-GPA antibodies in routine forensic examinations, potentially enhancing the accuracy of wound vitality assessments in criminal investigations.

Atrial fibrillation (AF) is the most common type of persistent arrhythmia. Although its incidence has been increasing, the pathogenesis of AF in stroke remains unclear. In this study, a total of 30 participants were recruited, including 10 controls, 10 patients with AF and 10 patients with AF and stroke (AF + STROKE). Differentially expressed genes (DEGs) were identified, and functional annotation of DEGs, comparative toxicogenomic database analysis associated with cardiovascular diseases, and predictions of miRNAs of hub genes were performed. Using RT‐qPCR, biological process and support vector machine neural networks, numerous DEGs were found to be related to AF. HBG1, SNCA and GYPB were found to be upregulated in the AF group. Higher expression of hub genes in AF and AF + STROKE groups was detected via RT‐PCR. Upon training the biological process neural network of SNCA and GYPB for HBG1, only small differences were detected. Based on the support vector machine, the predicted value of SNCA and GYPB for HBG1 was 0.9893. Expression of the hub genes of HBG1, SNCA and GYPB might therefore be significantly correlated to AF. These genes are involved in the incidence of AF complicated by stroke, and may serve as targets for early diagnosis and treatment.

Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.

Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175—GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175—GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)—Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5—BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.