The purpose of the proposed study is to investigate hereditary defects in band 3, the major anion transport protein. We have discovered two unique defects in band 3. One of the defects, """"""""slow migrating band3"""""""", is characterized by a band 3 molecule that migrates in gels as though it were Mr 98,500 instead of Mr approximately 95,000 as does normal band 3. Thus, it behaves as though there were an addition of Mr approximately 3,500 to the band 3 molecule. The other defect is """"""""fast-migrating band 3"""""""" which migrates in gels at Mr=92,000, as though there were a deletion approximately 2,000-3,000 rom the band 3 molecule. Cells from individuals with the latter defect have a significantly shortened lifespan and behave in many ways like old red blood cells.
The specific aims of this project are to (1) determine what functional defects are associated with band 3 structural defects; (2) define the structural defects in terms of band 3 domains and amino acid sequence; (3) determine whether the structural changes in band 3 affect its interaction with other membrane and cytoskeletal proteins and whether cytoskeletal proteins are normal; (40 determine the mode of inheritance of band 3 defects in kindreds; (5) characterize antibodies bound to red cells with band 3 anomalies; and (6) identify other defects in band 3 in the population. Band 3 will be characterized by gel electrophoresis before and after treatment with endoglycosidase F to remove carbohydrate groups. The number of IgG molecules per cell will be a determined using a DIDS binding assay. IgG binding studies will be performed. If there are abnormal amounts of IgG on red cells, IgG will be eluted and its specificity determined. Anion transport studies will be performed as a test of band 3 function, and ankyrin binding studies will used to test band 3 interaction with ankyrin. Band 3 proteins that are abnormal as determined by these assays will be analyzed by one and two dimensional peptide mapping and fast atom bombardment mass spectroscopy, and the amino acid sequence of the abnormal segment will be determined. Cytoskeletal proteins will be screened by gel electrophoresis and binding assays to determine whether their interactions with each other are normal. It is anticipated that these studies will provide insights into the structure and function of normal band 3 as well as into the molecular basis of some anemias.
|Poulin, J E; Cover, C; Gustafson, M R et al. (1996) Vitamin E prevents oxidative modification of brain and lymphocyte band 3 proteins during aging. Proc Natl Acad Sci U S A 93:5600-3|
|Kay, M M; Lake, D; Cover, C (1995) Band 3 and its peptides during aging, radiation exposure, and Alzheimer's disease: alterations and self-recognition. Adv Exp Med Biol 383:167-93|
|Kay, M M; Wyant, T; Goodman, J (1994) Autoantibodies to band 3 during aging and disease and aging interventions. Ann N Y Acad Sci 719:419-47|
|Kay, M M (1994) Regulatory autoantibody and cellular aging and removal. Adv Exp Med Biol 347:161-92|
|Kay, M M (1993) Generation of senescent cell antigen on old cells initiates IgG binding to a neoantigen. Cell Mol Biol (Noisy-le-grand) 39:131-53|
|Bruce, L J; Kay, M M; Lawrence, C et al. (1993) Band 3 HT, a human red-cell variant associated with acanthocytosis and increased anion transport, carries the mutation Pro-868-->Leu in the membrane domain of band 3. Biochem J 293 ( Pt 2):317-20|
|Kay, M M (1991) Band 3 in aging and neurological disease. Ann N Y Acad Sci 621:179-204|
|Kay, M M (1991) Drosophila to bacteriophage to erythrocyte: the erythrocyte as a model for molecular and membrane aging of terminally differentiated cells. Gerontology 37:5-32|
|Kay, M M; Hughes, J; Zagon, I et al. (1991) Brain membrane protein band 3 performs the same functions as erythrocyte band 3. Proc Natl Acad Sci U S A 88:2778-82|
|Kay, M M; Marchalonis, J J; Schluter, S F et al. (1991) Human erythrocyte aging: cellular and molecular biology. Transfus Med Rev 5:173-95|
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