The applicants note that the composition and organization of phospholipids across the bilayer membrane of the human erythrocyte are critical to the initiation and regulation of many cellular processes. While most membrane-related enzymatic and transport functions are associated with normal membrane lipid asymmetry, the redistribution of phosphatidylserine (PS) from its preferential location in the cell's inner leaflet to the outer leaflet results in a sequence of events that appears to be regulated by its display. For example, PS at the cell surface is critical to hemostasis, and may play a role in cell aging, membrane fusion, and the recognition and elimination of senescent and apoptotic cells. Although considerable progress has been made toward understanding aminophospholipid movement in red blood cells, the mechanism of lipid movement and its regulation are not understood, nor have the protein(s) involved in its control or recognition been identified. This proposal focuses on the organization, dynamics and spatial distribution of PS in the erythrocyte membrane and the mechanism by which reticuloendothelial cells recognize aged, PS-expressing cells. Particular emphasis is placed on identifying and characterizing the proteins that regulate the distribution and control the movement of PS in erythrocytes and PS binding proteins present in macrophage membranes. The proposed studies are based on results that have led to the concept that a 32 kDa membrane polypeptide, related to the Rh family of proteins, is involved in the generation and regulation of membrane lipid asymmetry. The main objectives of the application are to isolate, identify, and characterize PS binding proteins and the proteins responsible for transbilayer lipid movement. This will be done by a combination of techniques including isolation of transport protein from artificially- generated erythrocyte vesicles. Macrophage/monocyte PS binding proteins will be isolated by PS affinity reagents. The isolated proteins will be mapped and sequenced. Antibodies against PS binding proteins and the transporter as well as oligonucleotides generated from information of the sequenced protein will be used to screen appropriate cDNA expression libraries. Positive clones will be sequenced to identify the entire coding region of the transporter and PS binding proteins. The results of these studies will contribute toward understanding the regulatory processes that maintain specific transbilayer lipid distributions in human erythrocytes and mediate the recognition and ultimate elimination of aged, PS-expressing cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK041714-09
Application #
2838110
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Badman, David G
Project Start
1989-08-01
Project End
2000-11-30
Budget Start
1999-02-15
Budget End
2000-11-30
Support Year
9
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Texas MD Anderson Cancer Center
Department
Internal Medicine/Medicine
Type
Other Domestic Higher Education
DUNS #
001910777
City
Houston
State
TX
Country
United States
Zip Code
77030
Lee, A T; Balasubramanian, K; Schroit, A J (2000) beta(2)-glycoprotein I-dependent alterations in membrane properties. Biochim Biophys Acta 1509:475-84
Dombroski, D; Balasubramanian, K; Schroit, A J (2000) Phosphatidylserine expression on cell surfaces promotes antibody-dependent aggregation and thrombosis in beta2-glycoprotein I-immune mice. J Autoimmun 14:221-9
Diaz, C; Lee, A T; McConkey, D J et al. (1999) Phosphatidylserine externalization during differentiation-triggered apoptosis of erythroleukemic cells. Cell Death Differ 6:218-26
Diaz, C; Balasubramanian, K; Schroit, A J (1998) Synthesis of disulfide-containing phospholipid analogs for the preparation of head group-specific lipid antigens: generation of phosphatidylserine antibodies. Bioconjug Chem 9:250-4
Balasubramanian, K; Killion, J J; Schroit, A J (1998) Estimation of plasma beta-2-glycoprotein levels by competitive ELISA. Thromb Res 92:91-7
Balasubramanian, K; Schroit, A J (1998) Characterization of phosphatidylserine-dependent beta2-glycoprotein I macrophage interactions. Implications for apoptotic cell clearance by phagocytes. J Biol Chem 273:29272-7
Zwaal, R F; Schroit, A J (1997) Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89:1121-32
Balasubramanian, K; Chandra, J; Schroit, A J (1997) Immune clearance of phosphatidylserine-expressing cells by phagocytes. The role of beta2-glycoprotein I in macrophage recognition. J Biol Chem 272:31113-7
Diaz, C; Schroit, A J (1996) Role of translocases in the generation of phosphatidylserine asymmetry. J Membr Biol 151:1-9
Bruckheimer, E M; Schroit, A J (1996) Membrane phospholipid asymmetry: host response to the externalization of phosphatidylserine. J Leukoc Biol 59:784-8

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