Hereditary spherocytosis (HS) is a common, inherited hemolytic anemia in which defects of spectrin or the proteins that attach spectrin to the lipid bilayer (ankyrin, band 3 and protein 4.2) lead to spheroidal, osmotically fragile cells that are selectively trapped in the spleen. The prevailing theory is that HS is caused by local disconnection of the skeleton and bilayer, followed by vesiculation of the unsupported surface components. The mechanism of this effect is not well understood. One possibility is that the lipid bilayer is stabilized directly by interactions with the membrane skeleton. Alternatively, the lipids may interact with the multiple transmembrane helices of band 3, which would indirectly anchor them to the skeleton. We will test these two hypotheses using the techniques of targeted gene deletion and gene replacement in mouse embryonal stem (ES) cells. We will first determine the consequences of deleting band 3 or protein 4.2 in mice. Preliminary data show that band 3-I- mice have a severe spherocytic hemolytic anemia, but a surprisingly intact membrane skeleton, which fits best with the second hypothesis. We will evaluate this more directly by replacing band 3 with: (1) a band 3 derivative that lacks anion transport function, (2) a chimera of the glucose transporter attached to the cytoplasmic domain of band 3, (3) the isolated cytoplasmic domain of band 3 anchored to the membrane by a single transmembrane helix or by isoprenylation (4) the isolated membrane domain of band 3, and (5) a combination of the membrane and cytoplasmic domains, dually expressed but unattached to each other. The effects of each mutation on survival, organ function and pathology, red cell lifespan and morphology, red cell membrane and membrane skeletal composition and organization, membrane stability, and the function and mobility of band 3 will be investigated. Other aspects of membrane function will be measured in selected mutants, including ankyrin and/or spectrin rebinding to membrane vesicles, anion transport function, acid- base balance, and analysis of shed membrane vesicles.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK034083-18
Application #
2905311
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Badman, David G
Project Start
1983-07-01
Project End
2000-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
18
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Peters, Luanne L; Swearingen, Rebecca A; Andersen, Sabra G et al. (2004) Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency. Blood 103:3233-40
Eber, Stefan; Lux, Samuel E (2004) Hereditary spherocytosis--defects in proteins that connect the membrane skeleton to the lipid bilayer. Semin Hematol 41:118-41
Tse, W T; Tang, J; Jin, O et al. (2001) A new spectrin, beta IV, has a major truncated isoform that associates with promyelocytic leukemia protein nuclear bodies and the nuclear matrix. J Biol Chem 276:23974-85
Peters, L L; Jindel, H K; Gwynn, B et al. (1999) Mild spherocytosis and altered red cell ion transport in protein 4. 2-null mice. J Clin Invest 103:1527-37
Cho, M R; Eber, S W; Liu, S C et al. (1998) Regulation of band 3 rotational mobility by ankyrin in intact human red cells. Biochemistry 37:17828-35
Hoock, T C; Peters, L L; Lux, S E (1997) Isoforms of ankyrin-3 that lack the NH2-terminal repeats associate with mouse macrophage lysosomes. J Cell Biol 136:1059-70
Lu, F M; Lux, S E (1996) Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element. Proc Natl Acad Sci U S A 93:5663-7
Eber, S W; Gonzalez, J M; Lux, M L et al. (1996) Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis. Nat Genet 13:214-8
Peters, L L; Shivdasani, R A; Liu, S C et al. (1996) Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton. Cell 86:917-27
Shalev, O; Shinar, E; Lux, S E (1996) Isolated beta-globin chains reproduce, in normal red cell membranes, the defective binding of spectrin to alpha-thalassaemic membranes. Br J Haematol 94:273-8

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