Reticulocyte Enrichment and Culturing
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ERYTHROCYTE BIOLOGY AND ITS IMPACT ON PLASMODIUM VIVAX INVASION By EMILY SCHEETZ Submitted in Partial Fulfillment of the Requirements for the degree of Master of Science Department of Pathology CASE WESTERN RESERVE UNIVERSITY August, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Emily Scheetz candidate for the Master’s degree *. (signed) Mark Smith (chair of the committee) Chris King Clive Hamlin Peter Zimmerman (date) 7-8-2008 *We also certify that written approval has been obtained for any proprietary material contained therein. 1 TABLE OF CONTENTS LIST OF FIGURES………………………………………… 3 Abstract……………………………………………………... 4 Introduction…………………………………………………. 5 Chapter 1- Interaction of Malaria Parasites with human erythrocytes………………………………………………… 5 Erythrocyte Structural Proteins……………………. 5 Assembly of the Erythrocyte………………………. 8 Duffy Expression…………………………………… 10 Genetic Abnormalities of Erythrocytes…………… 16 Chapter 2- Erythrocyte Maturation……………………… 19 Chapter 3- Experimental Support……………………….. 22 Methods……………………………………………... 25 Results………………………………………………. 28 Discussion…………………………………………... 34 Future Directions…………………………………… 36 References………………………………………….. 39 2 LIST OF FIGURES 1. Figure 1: Erythrocyte Cytoskeleton Schematic……….. 10 2. Figure 2: Map of Duffy (-) Expression in Africa……….. 13 3. Table 1: Invasion Pathways of Plasmodium falciparum 14 4. Figure 3: Map of Overlap of Malaria with Abnormalities 15 5. Figure 4: Erythrocyte Abnormalities……………………. 16 6. Figure 5: Erythrocyte Protein Expression……………… 20 7. Figure 6: Enrichment of Cord Blood……………………. 29 8. Figure 7: Images of Enriched Cord Blood……………... 29 9. Figure 8: Culturing of Parasites…………………………. 31 10. Figure 9: CD71 Expression Through Time…………… 32 11. Figure 10: Glycophorin A Expression on Erythrocytes 33 12. Figure 11: Fy6 Expression on Erythrocytes………….. 33 13. Figure 12: Western Blot of Bound Protein……………. 34 3 Erythrocyte Biology and its Impact on Plasmodium vivax Invasion Abstract by EMILY SCHEETZ Malaria infection caused by Plasmodium species parasites is a world problem and the number of infections and deaths are staggering; 500 million and 1 million per year respectively. Although research on malaria has been extensive, a vaccine remains elusive. Characterization of the human erythrocyte proteins and their expression, and how Plasmodium species parasites exploit these proteins to gain entry into erythrocytes is an aspect of research that cannot be overlooked. In this paper, knowledge of erythrocyte biology, protein expression of erythrocytes, and genetic abnormalities of erythrocytes is combined to better understand the invasion biology of Plasmodium vivax malaria. This research may lay the groundwork for future insight into an in vitro culturing system for P. vivax as well as offer insight into vaccine development. 4 Introduction: The burden of malaria infection has impacted humans throughout the world for hundreds of years. Approximately 3.2 billion people are at risk for malaria [1] and vaccine development efforts have been unsuccessful thus far [2, 3]. Understanding the interaction between Plasmodium parasite species and erythrocytes is one of the most important aspects of vaccine development. The first step in this understanding is the functional role of the cytoskeleton and plasma membrane proteins of the erythrocyte. Expression of particular proteins, mainly the Duffy antigen receptor for chemokines (DARC or Duffy), may determine the invasion capacity of erythrocytes to Plasmodium vivax. Although factors affecting Duffy expression most certainly play a role in the variability of Duffy expression, very little is actually known. Genetic abnormalities of erythrocytes may be factors that affect Duffy expression. The localization of Duffy on the red cell surface may affect invasion of the parasite. Some preliminary data on Duffy expression has been assembled but a much deeper understanding must be reached. Compiling some well understood physical interactions of proteins within the human erythrocyte along with abnormalities and developing data on Duffy expression and parasite invasion, we seek to better understand the invasion pathways utilized by Plasmodium vivax by developing an in vitro culturing model and potentially be more informed about the development of a 5 vaccine against Plasmodium malaria by studying the expression of Duffy on the erythrocyte surface. Chapter 1- Red Blood Cell Biology and Abnormalities The interaction between the cytoskeleton and the plasma membrane of the erythrocyte is vital for the movement of erythrocytes into and out of the capillaries carrying oxygen and removing carbon dioxide from the bloodstream. This movement is also critical for the development of severe malaria and cerebral malaria. Each protein, and the interactions between proteins, contributes to the function or dysfunction of the erythrocyte. Basic knowledge about the constituents involved in erythrocyte function and parasite invasion of erythrocytes lays the ground work for filling in the gaps of scientific understanding. CYTOSKELETAL PROTEINS As in most cells, the cytoskeleton of erythrocytes is quite complex. The cytoskeleton is the network of proteins underlying and interacting with the cell membrane [4]. Multiple protein interactions must take place to keep the network intact and give the cell shape. The cytoskeleton determines the shape, integrity, and elasticity of erythrocytes [5-8]. Of all the cytoskeletal proteins, spectrin is the most abundant making up 75% of the erythrocyte cytoskeleton and is present at 100,000 copies per cell [9]. Because spectrin is the main component of the cytoskeleton it interacts with multiple proteins to form linkages [4]. Spectrin tetramerizes to form filaments which then interact with actin, 4.1, adducin, and 4.9 [10]. Other proteins such as glycophorins C and D and anion-exchanger 1 (band 3) associate with spectrin to 6 form interactions between the plasma membrane and the cytoskeleton [10]. Adaptor proteins such as ankyrin, 4.1, 4.2, and p55 also contribute to binding spectrin and creating the meshwork of the cytoskeleton [10]. Mutations in the spectrin gene lead to hemolytic anemias such as spherocytosis and elliptocytosis [11, 12] and knockouts of spectrin are lethal in nematodes, flies, and mice [13- 15]. RED BLOOD CELL SURFACE PROTEINS Red blood cell surface proteins play various roles in erythrocyte biology. The first important protein is band 3, also known as the anion exchanger. Named for its migration pattern when lysed erythrocytes were run on a gel, band 3 is one of the two most abundant integral proteins of the erythrocyte [16]. With one million copies per cell, band 3 makes up 30% of the total amount of membrane protein [9]. Band 3 is confined to cells of erythroid lineage and kidney cells, and is expressed on mature erythrocytes [17]. Band 3 is involved in membrane anion transport with its C-terminal domain mediating the exchange of chloride and bicarbonate anions, which increases the erythrocyte’s ability to carry CO2 from the tissues [16]. Another of its functions is to help with membrane stability by interacting with the cytoskeleton via Ankyrin-1 [18]. The cytoplasmic domain anchors the sub-membrane protein skeleton with the lipid bilayer by interacting with ankyrin, 4.1, and 4.2 [16]. A second surface protein expressed on erythrocytes is the transferrin receptor, also known as CD71. CD71 is expressed very early in the erythroid lineage maturation [19, 20] The transferrin receptor transports iron bound to 7 transferrin from the extracellular space inside the cell to form hemoglobin [21]. Without CD71, patients experience severe anemia [22]. There is variable expression on the surface of cells because of the varying needs for iron and hemoglobin synthesis [22]. Cells undergoing replication greatly up-regulate the expression of CD71 on their surface [22] and mature erythrocytes express very little CD71 [23]. CD71 is of interest because of its expression on reticulocytes and young cells. This fact is important when providing target cells to in vitro parasite cultures. Counting the number of young and old reticulocytes is done by flow cytometry detecting RNA and CD71 positivity respectively. The glycophorin family of proteins is another group expressed on the surface of erythrocytes. Glycophorins A-C play multiple roles on the surface of erythrocytes. They are the major sialoglycoproteins expressed on the surface [16, 19]. Sialic acids may be ligands for bacteria and viruses as well as parasites. Because the glycophorins give the erythrocyte its negative charge, they may prevent red cell aggregation in circulation and minimize cell-cell intereactions [16, 19]. Glycophorins also contribute to the glycocalyx which plays a role in protection from mechanical damage and microbial attack [16, 19]. Each of the previous proteins, as well as a multitude not described in detail here, interact to create the relationship between the cytoskeleton and plasma membrane. Cytoskeleton and Plasma Membrane Association Spectrin tetramers form a two-dimensional lattice by binding actin and other associated proteins forming a junctional complex. Dissociation and reassociation of the spectrin tetramers allows for the deformabilty of the 8 erythrocyte as it moves through the microvasculature [24]. The interaction between spectrin and the cytoplasmic surface of the plasma membrane controls the erythrocyte shape and elasticity. Spectrin interacts with ankyrin which subsequently