Defective anion transport activity of the abnormal band 3 in hereditary ovalocytic red blood cells.

Hereditary ovalocytosis is common in some areas of Melanesia and South East Asia where malaria is endemic. These red cells resist invasion by malarial parasites in vitro and ovalocytic individuals are less parasitized than normal. This has been attributed to the greater rigidity of ovalocytic red cells. It has been suggested that South East Asian ovalocytosis results from the heterozygous presence of an altered membrane anion transporter (band 3). We have used the polymerase chain reaction to clone the abnormal band 3 complementary DNA from an ovalocytic of Indian origin and found two changes from the normal protein: a point mutation (Lys 56----Glu) and the deletion of the sequence AFSPQVLAA (residues 400-408), but no evidence for an N-terminal extension. The deletion is also found in the abnormal band 3 of South East Asian ovalocytes and seems to be responsible for the unusual properties of the ovalocytic red cell. We show here that the membrane domain of the abnormal ovalocyte band 3 has a substantially altered structure and that the protein is defective in anion transport activity. The changed transport properties of the red cells may have a role in the reduced parasitaemia of ovalocytic individuals.

Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain.

Spectrin Tunis (Sp alpha I/78) is an alpha l domain variant that causes asymptomatic elliptocytosis in the heterozygote state. It is manifested by a reduction of spectrin dimer self-association and by the development of a major 78-Kd fragment at the expense of the alpha l 80-Kd fragment upon spectrin-limited digestion. Amino acid sequence analysis, following peptide transfer onto Immobilon membranes, showed that the 78-Kd fragment results from a sensitized cleavage after lysyl residue 10. Using a 13.5-kb genomic alpha-spectrin probe and the Xbal, Pvull, and Mspl polymorphic sites detected with this probe, we concluded that spectrin Tunis is associated with the + - + haplotype (in the above order). Twenty mer oligonucleotides, complementary to genomic segments from introns 2 and 3, respectively, were synthesized. We then performed DNA amplification and sequencing. In the two investigated carriers of spectrin Tunis, we found the C----T base substitution of the codon corresponding to position 35 of the alpha l domain (CGG----TGG; Arg----Trp). The mutation lies in the last part of an alpha helix that extends from residues 9 to 44 of partial repeat alpha 1' and is comparable with helix 3 of full repeats 1 to 5. The modified proteolytic site, located 25 amino acid residues upstream, occurs at the beginning of the helix.