Preliminary characterization of a structural defect in homozygous sickled cell alpha spectrin demonstrated by a rabbit autoantibody.
We have identified a rabbit autoantibody that strongly reacts with the core membrane skeleton of control red blood cells, and does not react with low- or high-density sickle cell core skeletons upon indirect immunofluorescence. Western blot analysis of red blood cell membrane proteins, utilizing this autoantibody, indicated no reactivity to any protein when SDS-PAGE was conducted in the presence of the reducing agent, dithiothreitol. However when SDS-PAGE was performed on control red blood cell membrane proteins separated in the absence of dithiothreitol, the autoantibody specifically reacted with a high molecular weight polypeptide (apparent Mr approximately equal to 310 kD) representing a DTT sensitive form of control alpha spectrin, which we refer to as alpha' spectrin. There was no staining of high density or low density sickle cell alpha or alpha' spectrin. This autoantibody should be an excellent tool for the fine mapping of structural change(s) in control vs. sickle cell alpha spectrin, and determination of whether the structural alteration effects spectrin dimer-tetramer interconversion and/or the spectrin-actin interaction. The modification in alpha spectrin, detected by this antibody, is very specific for homozygous SS alpha spectrin because sickle cell beta+ thalassemic alpha spectrin and sickle cell trait alpha spectrin react intensely with the autoantibody.
Monteiro
C A
CA
Department of Structural and Cellular Biology and USA Comprehensive Sickle Cell Center, University of South Alabama College of Medicine, Mobile 36688, USA.
Gibson
X
X
Shartava
A
A
Goodman
S R
SR
D000755:Anemia, Sickle Cell / Q000097:blood*; D000818:Animals; D001323:Autoantibodies / Q000097:blood; D015153:Blotting, Western; D004229:Dithiothreitol / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000276:immunology; D004913:Erythrocytes, Abnormal / Q000737:chemistry*; D006720:Homozygote; D006801:Humans; D008565:Membrane Proteins / Q000097:blood / Q000276:immunology; D011817:Rabbits / Q000276:immunology*; D012967:Sodium Dodecyl Sulfate; D013049:Spectrin / Q000032:analysis* / Q000276:immunology*
D001323:Autoantibodies; D008565:Membrane Proteins; D013049:Spectrin; D012967:Sodium Dodecyl Sulfate; D004229:Dithiothreitol