Dr. Goodman's laboratory has recently identified a molecular defect in the erythrocyte membrane skeleton of two of four hereditary spherocytosis (HS) kindreds under investigation. In these HS kindreds (Type I HS), a defective spectrin molecule leads to an altered spectrin-protein 4.1 interaction, which in turn leads to a defective spectrin-protein 4.1-actin ternary complex. As this defect does not correlate with red cell age in the circulation or splenic function, but does correlate with the defective gene dose in this autosomal dominant disorder it may be the primary defect causing HS in these kindreds. In the proposed studies the other two kindreds (Type II HS) as well as newly available HS kindreds will be screened for a defect in their membrane skeletons by functional assays performed on the purified proteins, as well as one and two dimensional peptide mapping analysis of skeletal proteins. The Type I defect will be identified by a series of experiments including [1] isolation of the defective HS gene product by affinity chromatography on a protein 4.1-sepharose column, [2] isolation of the defective 74,000 Mr Beta-4 domain from the defective HS gene product and normal spectrin, [3] protein 4.1 binding analysis and peptide mapping analysis on the 74,000 dalton tryptic fragment of the Beta subunit confirming the localization of the defect, and [4] amino acid sequence analysis on the Type I HS and normal 74,000 Mr Beta-4 domain. These studies should lead to the identification of the exact amino acid substitution (or less likely post translational modification) causing the Type I HS membrane skeletal disorder. We will also study the association of protein 4.1 with it's membrane attachment site so that this new functional assay can be used to screen HS subjects for a potential defect in this interaction, and towards our goal of identifying the high affinity protein 4.1 membrane attachment protein. These studies should lead to major advances in our understanding of the molecular basis of HS, as well as elucidating the functional interactions of protein 4.1 in the normal erythrocyte membrane skeleton.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL026059-06
Application #
3338431
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1983-08-01
Project End
1988-07-31
Budget Start
1985-08-01
Budget End
1986-07-31
Support Year
6
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Riederer, B M; Goodman, S R (1990) Association of brain spectrin isoforms with microtubules. FEBS Lett 277:49-52
Karinch, A M; Zimmer, W E; Goodman, S R (1990) The identification and sequence of the actin-binding domain of human red blood cell beta-spectrin. J Biol Chem 265:11833-40
Riederer, B M; Lopresti, L L; Krebs, K E et al. (1988) Brain spectrin(240/235) and brain spectrin(240/235E): conservation of structure and location within mammalian neural tissue. Brain Res Bull 21:607-16
Riederer, B M; Zagon, I S; Goodman, S R (1987) Brain spectrin(240/235) and brain spectrin(240/235E): differential expression during mouse brain development. J Neurosci 7:864-74
Krebs, K E; Zagon, I S; Sihag, R et al. (1987) Brain protein 4.1 subtypes: a working hypothesis. Bioessays 6:274-9
Krebs, K E; Prouty, S M; Zagon, I S et al. (1987) Structural and functional relationship of red blood cell protein 4.1 to synapsin I. Am J Physiol 253:C500-5
Goodman, S R; Zagon, I S; Riederer, B M (1987) Spectrin isoforms in mammalian brain. Brain Res Bull 18:787-92
Krebs, K E; Zagon, I S; Goodman, S R (1987) Amelin: a 4.1-related spectrin-binding protein found in neuronal cell bodies and dendrites. J Neurosci 7:3907-14
Riederer, B M; Goodman, S R (1987) Immunological detection of high molecular weight proteins by gel and blot overlay. Brain Res Bull 19:715-22
Krebs, K E; Zagon, I S; Goodman, S R (1987) Amelin and synapsin I are 4.1 related spectrin binding proteins in brain. Brain Res Bull 18:793-8

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