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Membrane Proteome Profiling of Erythroid Cell Lineages: A Valuable Resource for Advancing Plasmodium vivax Research

ABSTRACT: Background: Erythropoiesis is an intricate maturation process culminating in the production of erythrocytes. As cells progress through erythropoiesis, they undergo surface loss, organelle expulsion, size reduction, and dynamic removal of membrane proteins, refining their structure for specialized physiological functions. This orchestrated process is essential for generating mature erythrocytes capable of efficient gas exchange in the circulatory system. The membrane composition of red blood cells, comprising lipid layers and embedded proteins that play a fundamental role in cellular stability, flexibility, and host interactions. Investigating the membrane proteome becomes particularly relevant in the context of infectious diseases, such as malaria caused by Plasmodium. These proteins critically interact with parasite ligands, allowing entry and progression through various life cycle stages within host cells. Among Plasmodium species, P. vivax exhibits a specific tropism for invading reticulocytes, posing challenges for in vitro continue cultures development due to their limited availability and rapid differentiation. To address this, we studied hematopoietic cell lines, JK-1 and BEL-A2, as potential alternatives to reticulocytes for studying P. vivax biology. Using a Data Independent Acquisition (DIA) LC-MS/MS approach, we characterize and compare the membrane proteomes of reticulocytes, erythrocytes, and these cell lines. Results: Our results reveal distinct abundance patterns in transmembrane proteins, skeletal proteins, and membrane transporters during erythropoiesis. Additionally, we identified 6 potential receptor candidates for P. vivax merozoites ligands, shedding light on the intricate host-parasite interactions. Furthermore, we detected the loss of membrane proteins during erythroblast enucleation, providing insights into the cellular processes governing this transition. Conclusions: This study significantly contributes to our understanding of erythropoiesis, offering valuable insights for P. vivax malaria research and in vitro culture system development. The comprehensive analysis of membrane proteomes in different cell stages establishes a foundation for future investigations into cellular homeostasis, disease mechanisms, and potential therapeutic targets.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Manuel Alfonso Patarroyo Markus Kalkum

PROVIDER: MSV000093438 | MassIVE | Mon Nov 20 12:56:00 GMT 2023

REPOSITORIES: MassIVE

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Project description:Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterised by the accumulation of infected erythrocytes in the microvasculature of the brain, due to parasite adhesins on the surface of infected erythrocytes binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We used the Human Brain Endothelial Cell line HBEC-5i to identify the malaria parasite ligands responsible for binding to human brain endothelial cells. Three P. falciparum strains (HB3, 3D7 and IT/FCR3) were selected for binding to HBEC5i and the whole transcriptome of selected and unselected parasites was analysed using a variant surface antigen-supplemented microarray chip. After selection, the only highly upregulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7 and ITvar19), that showed 11 to >100-fold higher transcription levels in selected parasites. These genes are highly diverse in sequence, but do however show strong similarities in PfEMP1 architecture. Antibodies raised to the HB3var3 variant recognized the surface of infected erythrocytes and abolished the binding of infected erythrocytes to brain endothelial cells. The subset of Group A PfEMP1 variants identified here provides a new target for interventions to treat or prevent cerebral malaria. Unselected Plasmodium falciparum parasites (Uns) only poorly cytoadhere to human brain endothelial cells (HBEC-5i). Plasmodium falciparum HB3, 3D7 and IT/FCR3 strains were selected for binding to HBEC-5i (HB3-HBEC1, HB3-HBEC2, HB3-HBEC-TNF, 3D7-HBEC and IT-HBEC). HBEC-selected and unselected cultures were tightly synchronised before a timecourse experiment was performed. 6 samples, named time points 1 to 6, were taken every 8 hours. 12μg of RNA from the unselected parasites (HB3-Uns, 3D7-Uns or IT-Uns) at each of the 6 time points was combined together to form the reference pool. The pool and 12μg of each individual time point sample from both selected and unselectedparasites were then used for cDNA synthesis. For HB3-HBEC1 (pilot experiment), each HB3-HBEC1 time point (pilot experiment) was hybridised directly with HB3-Uns1 time points. For microarray hybridizations, each cDNA sample was coupled to Cy5 (red dye) while Cy3 (green dye) was added to the pool. Cy5-labelled time point samples were mixed with the same amount of Cy3-labelled pool sample. The solution was loaded on a microarray slide and hybridized for 14–16 h.

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Membrane Proteome Profiling of Erythroid Cell Lineages: A Valuable Resource for Advancing Plasmodium vivax Research

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