Sickle Cell Disease is a multi-system disorder, occurring when the sickle hemoglobin gene is inherited from both parents, causing disturbances in red blood cell transit into the spleen and liver vasculature. Although sickle cell anemia remains a significant cause of mortality in Sub-Saharan Africa, sickle hemoglobin can be advantageous in some cases.
Today, there are 250 million people carrying red blood cells (RBCs) with sickle hemoglobin but not necessarily suffering from the disease. On the contrary, the sickle cell trait helps them develop resistance against malaria.
This phenomenon has been attributed to the phagocytosis of infected RBCs with sickle hemoglobin, thus eliminating the infection before the parasite P. falciparum can infiltrate the circulation. However, the mechanism by which sickle RBCs are recognized by the body’s immune system has not been thoroughly understood.
Researchers from the University of Aberdeen, in collaboration with research laboratories across the UK, hypothesized that enhanced oxidative stress provoked RBCs to express high-mannose glycans on the surface, and these glycans were recognizable by mannose receptors on spleen macrophage surfaces, facilitating RBC destruction (i.e., hemolysis) through phagocytosis (1).
The researchers first investigated the type of surface ligands highly expressed on RBC surfaces and their active sites using flow cytometry with 7 carbohydrate-specific plant lectins from Vector Laboratories and 1 other plant lectin. Carbo-Free Blocking Solution helped eliminate false staining due to possible contamination. Then, they aimed to correlate high-mannose N-glycan expression with extravascular hemolysis in anemia patients and sickle hemoglobin carriers.
To unravel the underlying mechanism of high-mannose N-glycan expression, the researchers assessed the lectin binding levels of healthy RBCs upon increased oxidative stress. Western blots probed with Galanthus nivalis Agglutinin (GNA) lectin revealed protein structures accompanying the migration and accumulation of high-mannose glycans.
Finally, the researchers assessed how the altered RBC glycan expressions could provide protection against P. falciparum infection, comparing infection-induced glycan expression in both healthy donors and those with sickle cell traits.
The macrophages display surface lectins that recognize and capture RBCs with sickle cell traits (2). That’s why it was first necessary to identify lectins that mainly recognize diseased blood cells. In a panel of plant lectins, the RBCs with sickle hemoglobin were bound to GNA, with specificity for the terminal mannose residues of N-glycans. Because the lectin was fluorescently labeled, red staining could be detected as it bound to the RBCs with sickle hemoglobin; no staining was recorded in healthy RBCs.
If the sickle cell trait is detected by interactions between mannoses on RBC surface glycans and GNA lectin, these interactions should correlate with the level of extravascular hemolysis and the severity of anemia. Indeed, samples from patients with a homozygous sickle hemoglobin profile (i.e., HbSS) displayed significantly higher binding than both the heterozygous samples (HbAS), associated with mild anemia, and healthy patient samples (HbAA). Furthermore, the hemolysis cell marker measurements, such as an apoptotic phagocytosis marker, were in line with high- mannose N-glycan expression and GNA lectin binding.
Next, the researchers delved deeper into the factors behind induced surface mannose expression and its impact on extravascular hemolysis. Since intracellular oxidative stress is a well-known hallmark of sickle cell disease (3), they hypothesized that it should affect glycan expression on RBC surfaces. To monitor the altered mannose expression under immunofluorescence microscopy, GNA lectin was combined with streptavidin (red). When healthy RBCs were exposed to an oxidizing agent, they exhibited a higher level of high-mannose N-glycans.
In addition, a panel of recombinant mammalian C-type lectin fusion proteins revealed the mannose receptor (MR, CD206) to be the site of recognition by macrophage lectins. The fact that mannose receptor–blocking antibodies inhibited phagocytosis of diseased RBCs confirmed the role of this receptor in RBC recognition by phagocytes.
The next step was to identify specific proteins in the high-mannose N-glycan structures, so the researchers used western blots probed with GNA lectin. The strongest lectin binding was observed at a molecular weight corresponding to membrane skeletal proteins α-spectrin and β-spectrin. Further western blot analysis confirmed that the spectrin-containing complexes were N-glycosylated, and these complexes had low molecular weights and unconventional glycated forms. These membrane skeletal proteins are associated with high-mannose N-glycans, which function as an eat-me signal and are associated with two other ligands. Because of their structural features, their complete identification was not possible, but the researchers were still able to confirm that these complexes could form aggregates on RBC surfaces to enable mannose exposure.
After confirming the mechanism through which RBCs experienced hemolysis upon oxidative stress, the researchers looked at its implications for malaria infection with P. falciparum. It is evident that malaria patients with sickle cell traits exhibited higher clearance of infection than those with healthy wild-type (no sickle cell trait) RBCs (4). The question was whether the mannose recognition mechanism played a role in malaria resistance in sickle cell traits.
Infection of healthy RBCs and sickle hemoglobin RBCs resulted in increased high-mannose N-glycan exposure and, subsequently, higher GNA lectin binding. It was noted that these N-glycans formed at earlier stages of malaria (and in higher amounts) in sickle RBCs than in healthy RBCs. This confirmed that N-glycan-mediated malaria resistance was much higher in sickle cell trait.
The major obstacle in malarial infection clearance is the expression of the antigen PfEMP1—responsible for the cytoadherence of the infected cell to the vascular endothelium—by the parasite. As expected, donors with sickle cell trait showed much lower amounts of this antigen than the control group, meaning sickle cell RBCs were cleared much more easily when infected with P. falciparum.
The relation between sickle hemoglobin and malaria resistance has been a long-standing mystery, one that was never properly addressed on a molecular level. However, this study provides an explanation, unraveling the interactions between RBC surface glycans and the surface receptors of phagocytes. More specifically, it found that RBCs with sickle hemoglobin have induced oxidative stress, prompting them to express high-mannose N-glycans on the RBC surface. These glycans are accompanied by covalently linked spectrin-containing complexes that are strongly recognized by phagocytic mannose receptors.
In the context of malaria infection, RBCs with sickle hemoglobin are easier to eliminate before they can infiltrate the blood flow. In other words, the sickle hemoglobin prevents the infected cells from adhering to the vasculature, making them readily available for phagocyte recognition.
This study could substantiate the idea that the sickle-cell mutation was naturally selected in Sub-Saharan Africa to provide a survival advantage against malaria. More importantly, a better understanding of this mechanism could aid in the development of novel therapies for malaria.
For more information on Lectin application and usage, check out our extensive Lectins Application & Resource Guide. This content rich educational booklet provides insight into how lectins are applied in biological sciences. With a focus on plant derived lectins, the guide showcases the flexibility and utilization of lectins and lectin conjugates in established lab applications.