MOLECULAR CHARACTERIZATION OF THE BAND-3 PROTEIN FROM SOUTHEAST-ASIANOVALOCYTES

Southeast Asian ovalocytosis (SAO) is a hereditary form of elliptocyto sis resulting in rigid, oval-shaped erythrocytes resistant to invasion by malaria parasites. The molecular defect is due to deletion of codo ns 400-408, encoding a 9-amino-acid sequence located at the boundary b etween the cytosol and the first transmembrane segment in Band 3, the erythrocyte anion transport protein. We have carried out an extensive characterization of Band 3 isolated from SAO erythrocytes which contai n about 50% mutant Band 3. A slightly higher proportion of Band 3 in S AO erythrocytes was left associated with the cytoskeleton after extrac tion of ghost membranes with non-ionic detergents. Size exclusion high performance liquid chromatography analysis showed that SAO Band 3 con tained a higher proportion of tetramers relative to dimers (50% tetram er) than normal Band 3 (33% tetramer). The circular dichroism spectrum of Band 3 from SAO erythrocytes was very similar to the spectrum for normal Band 3. Enzymatic deglycosylation and tomato lectin binding sho wed that SAO Band 3 lacked the polylactosaminyl oligosaccharide found on normal Band 3. SAO Band 3 was unable to bind the anion transport in hibitor 4-benzamido-4'-aminostilbene-2,2'-disulfonate, suggesting a dr amatic alteration in the inhibitor binding site. In conclusion, deleti on of 9 amino acids from Band 3 on the cytosolic side of the membrane affects the properties (glycosylation and inhibitor binding) of Band 3 on the opposite side of the membrane without dramatic changes in the secondary and quaternary structure of the protein.