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Molecular characterization of the band 3 protein from Southeast Asian ovalocytes
V E Sarabia1, J R Casey, R A Reithmeier
1Department of Medicine, University of Toronto, Ontario, Canada.
Insights
Southeast Asian ovalocytosis (SAO) involves a Band 3 protein defect, making red blood cells rigid and malaria-resistant. This defect alters protein glycosylation and inhibitor binding without changing its core structure.
Area of Science:
- Genetics
- Hematology
- Biochemistry
Background:
- Southeast Asian ovalocytosis (SAO) is an inherited elliptocytosis.
- SAO erythrocytes exhibit resistance to malaria parasite invasion.
- The condition stems from a specific deletion in the Band 3 protein.
Purpose of the Study:
- To characterize Band 3 protein in SAO erythrocytes.
- To investigate the structural and functional consequences of the Band 3 mutation.
Main Methods:
- Isolation and characterization of Band 3 from SAO erythrocytes.
- Analysis of protein association with the cytoskeleton.
- Size exclusion high-performance liquid chromatography (SE-HPLC).
- Circular dichroism spectroscopy.
- Enzymatic deglycosylation and lectin binding assays.
- Anion transport inhibitor binding studies.
Main Results:
- SAO Band 3 showed increased association with the cytoskeleton.
- SE-HPLC revealed a higher tetramer-to-dimer ratio in SAO Band 3.
- SAO Band 3 lacked specific polylactosaminyl oligosaccharides.
- The mutation abolished binding of the anion transport inhibitor 4-benzamido-4'-aminostilbene-2,2'-disulfonate.
- Circular dichroism spectra indicated minimal changes in secondary structure.
Conclusions:
- The 9-amino acid deletion in Band 3 affects glycosylation and inhibitor binding.
- These changes occur on the opposite side of the membrane from the mutation.
- SAO Band 3's structural integrity (secondary and quaternary) remains largely intact.
Abstract:
Southeast Asian ovalocytosis (SAO) is a hereditary form of elliptocytosis resulting in rigid, oval-shaped erythrocytes resistant to invasion by malaria parasites. The molecular defect is due to deletion of codons 400-408, encoding a 9-amino-acid sequence located at the boundary between 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 contain about 50% mutant Band 3. A slightly higher proportion of Band 3 in SAO erythrocytes was left associated with the cytoskeleton after extraction of ghost membranes with non-ionic detergents. Size exclusion high performance liquid chromatography analysis showed that SAO Band 3 contained a higher proportion of tetramers relative to dimers (50% tetramer) 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 showed that SAO Band 3 lacked the polylactosaminyl oligosaccharide found on normal Band 3. SAO Band 3 was unable to bind the anion transport inhibitor 4-benzamido-4'-aminostilbene-2,2'-disulfonate, suggesting a dramatic alteration in the inhibitor binding site. In conclusion, deletion 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.