The membrane of the red blood cell (RBC) contains a great variety of phospholipid (PL ) molecular species that undergo continuous structural/molecular and organizational remodeling during the life span of the cell. Since de novo synthesis of lipids does not occur in mature erythrocytes, the cell is dependent on plasma for lipid substrates. Differences between plasma and red cell PLs indicate that the red cell must be capable of selectively remodeling PLs. Since changes in PL molecular species and organization alter red cell structure and function, we hypothesize that proper maintenance of membrane PL composition and organization (PL homeostasis) is tightly regulated. To pursue this hypothesis, we will: 1) characterize the molecular regulation of PL deacylation/reacylation reactions required for PL remodeling, 2) investigate the role of PL homeostasis in red cell membrane remodeling during cell maturation, and 3) investigate the mechanisms responsible for loss of phosphatidylserine asymmetry in the lipid bilayer and the consequences of this loss. The mechanisms that regulate acylation, deacylation, and molecular species composition of membrane PL will be investigated by studying the turnover of different fatty acids within specific PL molecular species, and characterizing two key enzymes involved in the deacylation/reacylation process. Using molecular biology techniques, we plan to purify a 29 kDa phospholipase A2 (PLA2) that deacylates PLs and, using a novel technique involving the synthesis of a lipid membrane around the active enzyme, purify the membrane bound 42 kDa lysoPL-acyltransferase (LAT) that reacylates PLs. Biochemical, molecular and immunologic techniques will be used to characterize these enzymes and to determine if they represent a member of a family of enzymes with specificity for a given PL molecular species. The role of these two enzymes in PL remodeling will be investigated using purified red cell membranes, artificial membrane preparations containing specific PL substrates, and lipid monolayer systems. To investigate our hypothesis that alterations in PL homeostasis contribute to membrane remodeling during cell maturation, studies of reticulocytes and neonatal red cells will be performed. PL homeostasis in plasma and intracellular membranes of reticulocytes will be studied by using transferrin receptor based methods to separate the plasma membrane from intracellular vesicles. Studies of neonatal red cells will provide the opportunity to determine if there are developmental changes in the cells' ability to remodel PLs and to determine if there are unique mechanisms for PL remodeling given the unusual lipid characteristics of neonatal plasma compared to adult plasma. Mechanisms responsible for, and consequences related to, loss of PL asymmetry will be investigated using annexin V. This probe will enable us to use FACscan and fluorescence microscopy to identify cells with PS on their surface, study the distribution of PS in the membrane of individual cells and investigate the potential pathophysiologic effects of abnormalities in PS organization. In summary. our research program should a) provide new information on the structure and function of two key enzymes required for PL homeostasis, b) determine if alterations in PL homeostasis contribute to reticulocyte and neonatal red cell membrane remodeling during red cell maturation and c) provide new information on the mechanisms responsible for loss of PL asymmetry and its potential physiologic effects in human erythrocytes.
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