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Phospholipid molecular species turnover

More than 250 different glycerophospholipid and sphingomyelin molecular species make up the structural backbone of the red cell lipid bilayer. While the overall composition and organization is well maintained during the life of the cell, this is a very dynamic system in which lipid molecules are continuously renewed and rapidly move in the plane of each layer, as well as across the bilayer. Our overall research goal is to better understand the underlying mechanisms that keep this dynamic integrity intact, and the consequences that alterations of this structure have for red cell pathology. Whereas the red cell lacks de novo lipid synthesis, the phospholipids in the plasma membrane are continuously renewed by a deacylation/reacylation mechanism (the Lands Pathway). Long chain acyl-CoA synthetase (LACS, EC plays a key role in this pathway. We have cloned, sequenced, and expressed full-length cDNAs encoding two forms of acyl-CoA synthetase from a K-562 human erythroleukemic cell line . The first form, named Long-chain Acyl-CoA Synthetase 5 (LACS5), was found to be a novel, unreported, human acyl-CoA synthetase, while the second form (66% identical to LACS5) was 97% identical to human liver LACS1. The novel LACS5 gene encodes a highly expressed 2.9 kilobase (kb) and 6.3 kb mRNA transcript in human red cell precursors. However, transcripts are virtually absent in human heart, kidney, liver, lung, pancreas, spleen, and skeletal muscle. Antibodies directed against LACS5 cross-reacted with red cell membranes indicating that LACS5 plays an important role in plasma membrane fatty acid utilization. We expressed it as an active enzyme in E-coli. Interestingly, LACS5 is also highly expressed as a 2.9 kb and 9.6 kb mRNA transcript in human brain, and distinctly different (65% sequence homology) from LACS4, a form of this enzyme previously reported to be present in the human brain. The 78 kDa expressed LACS5 protein uses long chain fatty acids such as palmitic acid, oleic acid, and arachidonic acid as substrates.

It has recently been reported that fatty acid utilization may be different in patients with psychiatric disorders such as schizophrenia. Our current studies show that the red cell phospholipid molecular species are different in schizophrenia. We hypothesize that an altered LACS5 activity may be responsible. Studies in blood samples to characterize red cell acyl CoA synthetase activity in combination with LACS5 sequence identity may shed light on the altered utilization of fatty acids in the brain of these patients.

Phospholipid organization

In addition to the complexity due to the large variety of molecules with different properties, the phospholipids are organized in a specific and asymmetric fashion across the phospholipid bilayer.

FIGURE 1: Phospholipid asymmetry

In steady state, the choline containing phospholipids, sphingomyelin (SM) and phosphatidylcholine (PC) are mainly found on the outside of the bilayer while the aminophospholipids are mainly (phosphatidylethanolamine, PE), or exclusively (phosphatidylserine, PS), found in the inner monolayer. This organization is maintained by an active set of transport systems that "flip" and "flop" phospholipids across the membrane. The flipase actively transports aminophospholipids from the outer to the inner monolayer, while the scramblase, when activated, moves all phospholipids in both directions, thereby scrambling the phospholipid distribution. PS exposed on the surface forms a docking site for hemostatic factors such as the prothrombinase complex (factor Xa, Va and II). In addition, PS is recognized by macrophages and interacts with proteins such as annexin V.

We study the mechanisms that underlie this asymmetric distribution and have developed an efficient way to determine the loss of phospholipid asymmetry in subpopulations of red cells. PS exposure requires inhibition of the flipase as well as activation of the scrambling process, possibly by elevation of intracellular calcium. Our data show that indeed the flipase is inhibited in PS-exposing RBC. On the other hand, using the fluorescent calcium indicator Fluo3, we find that calcium is not permanently elevated in these RBC, although it can not be excluded that transient increases in intracellular calcium could cause episodes of phospholipid scrambling. Our recent data show that activation of protein kinase C (PKC) by phorbol ester causes immediate PS exposure in a subpopulation of RBC in presence of extracellular calcium. However, increased cytosolic calcium was only found in a small subpopulation of PS-exposing cells. Inhibition of PKC with either calphostin C or chelerythrine chloride diminished both the formation of PS-exposing cells and calcium influx. Calphostin C also inhibited the calcium-activated membrane scrambling induced in vitro with calcium ionophore A23187. Our data indicate that calcium influx, although essential for scrambling, can be uncoupled from PS exposure, and that PKC is implicated in the mechanism of membrane phospholipid scrambling. While PS exposure has been recognized as an early step in apoptosis, the mechanisms that lead to this exposure have not been elucidated. We feel that a better understanding of the exposure of PS in RBC will also render information on the mechanisms of PS exposure in the plasma membranes of other cell types, and as such shed light on an important step in apoptosis.


Many humans carry mutations in the globin genes. Globin genes determine the structure of hemoglobin, the oxygen-carrying protein of the red cell. Most prevalent are mutations that lead to disease states such as thalassemia or sickle cell anemia, affecting many millions of individuals worldwide. The relative protective effect against malaria of the genes that lead to altered hemoglobin may be the underlying reason for the high prevalence of these mutations in the human population. Our laboratory studies these disorders in collaboration with the hematology department of Children’s Hospital Oakland.

Despite the fact that the underlying mutations of the hemoglobinopathies are in the structural or regulatory genes for globin, the red cells in these disorders are often characterized by abnormal membranes. We have shown that in both sickle cell anemia and thalassemia, subpopulations of red cells exist that have an abnormal lipid organization, exposing phosphatidylserine. This exposure has very significant physiological consequences, leading to imbalance in hemostasis as well as altered cell-cell interactions, and recognition and removal of these red cells that contributes to the anemia. Our studies indicate that these abnormalities in membrane organization are related to high levels of apoptosis (programmed cell death) in red cell precursors, as well as to acquired defects during the life of the adult red cell. In our studies, murine models of sickle cell anemia and thalassemia have been very useful. Highlights of recent studies are described in the next section.TopSickle RBC, intermittent hypoxia, and vascular pathologyThe unique oxygen-sensing capacity of the sickle red cell defines sickle cell disease pathology as resulting from the polymerization of sickle hemoglobin under low partial oxygen pressure. Our long-term goal is to understand how intermittent hypoxia changes the red cell membrane and its interactions with the vascular endothelium, using a simple working hypothesis as indicated in the figure.

FIGURE 2: Hypoxia and sickle cell membrane organization

The mutation in hemoglobin leads to the exposure of Phosphatidylserine (PS) on the surface of the red cell (RBC) under low oxygen. This leads to a number of physiologic consequences including altered red cell endothelial interactions.

In sickle cell disease (SCD), an abnormal subpopulation of PS-exposing red blood cells is found in the circulation. As determined with fluorescent annexin V, this population averages 2% in human patients and 4% in transgenic sickle mice that exclusively make human sickle hemoglobin. Most PS-exposing sickle cells are found in the low (d<1.08) or high (d>1.12) density red cell fractions. Analysis of these fractions showed PS-exposing subpopulations in mature cells, as well as in RNA-containing, or transferrin receptor expressing young erythrocytes. Increased PS exposure was also found on erythroid precursors in murine bone marrow and spleen. Our studies on the survival of sickle murine RBC using in vivo biotinylation demonstrate that PS-exposing cells are prematurely removed from the population, which may contribute to the shortened survival of RBC in SCD. Together, our data suggest that PS-exposing RBC are continuously formed, followed by rapid removal from the circulation. However, the removal is not efficient enough to avoid an increased presence of these cells at steady state, which in turn will have consequences for SCD pathophysiology, including an imbalance in hemostasis and increased cell-cell interaction. Our studies on normal cells have shown that PS exposure requires inhibition of the flipase as well as activation of the scrambling process, possibly by elevation of intracellular calcium. Indeed the flipase is inhibited in the PS-exposing sickle cells. On the other hand, calcium is not permanently elevated in PS-exposing sickle cells, although it can not be excluded that transient sickling-induced increases in intracellular calcium could cause episodes of phospholipid scrambling. Our data suggest that the increased PKC activation shown in sickle cells during sickling may be the underlying reason for the presence of PS-exposing subpopulations. During the process of PS exposure, RBC also generate lysophosphatidic acid (LPA), a powerful lipid mediator. We have shown that PS-exposing red cells can activate endothelial cells through an LPA-mediated process, exposing the underlying matrix and causing RBC-binding to thrombospondin in a PS-mediated way. We have reported that sickle cell patients show dramatically increased levels of the inflammatory mediator secretory phospholipase A2 (sPLA2) in their serum during episodes of acute chest syndrome. The increase in the levels of this enzyme, which we have proven to be of the type IIa, is predicting the onset of acute chest syndrome , and also seems to play a key role in the actual damage to the lung. We have shown that this enzyme can interact with abnormal apoptotic or necrotic, PS-exposing membranes, thereby generating products (including LPA) that affect normal endothelial function. In addition to this important inflammatory mediator, other soluble compounds can modulate cellular interactions in sickle cell disease. Our recent studies show that we can manipulate nitric oxide (NO) levels by L-Arg supplementation during vaso occlusive crisis and acute chest syndrome. We pose that nitric oxide and related compounds generated by the endothelium under hypoxia will affect the RBC endothelial cell interaction, depending on the hemoglobin concentration and type (HbS or HbF). In short, our studies have provided the basis for the working hypothesis as indicated in figure 2, and have resulted in a number of translational studies.

Translational studies and the Red Cell Resource Laboratory

Our laboratory has set up a number of important and unique assays that have been helpful for the characterization of red cell abnormalities, including the measurement of red cell deformability, density distribution, oxygen carrying capacity, and membrane surface properties. In addition, we have established several analytical techniques for blood components important for clinical studies performed in collaboration with the hematology, emergency and pulmonary departments at Children’s Hospital Oakland. Our current translational studies include the characterization of red cells in correlation with bone marrow transplantation in hemoglobinopathies (with Dr. Walters, Hematology/Oncology), the role of sPLA2 in acute chest syndrome in sickle cell disease (with Drs. Styles and Vichinsky, Hematology/Oncology), the role of arginine, the precursor of nitric oxide, in vaso-occlusive crisis and acute chest syndrome in sickle cell disease (with Dr. Morris, Emergency and Dr. Styles, Hematology/Oncology), and the characterization and chemotherapeutical treatment of hemoglobin E-beta thalassemia (with Dr. Singer, Hematology/Oncology).


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