FUNCTIONAL FACTORS IN THE RED-CELL MEMBRANE - INTERACTIONS BETWEEN THE MEMBRANE AND ITS UNDERLYING SKELETON

Recent studies involving two abnormal red cell phenotypes (South-east Asian ovalocytosis and Leach phenotype) provide novel information concerning the nature and significance of interactions of both the anion transport protein AE-1 (syn. band 3) and Glycophorins C and D with the underlying skeleton. The location of Wr(a) and Di(a) blood group antigens to mutations on AE-1 at residues 658 and 854 respectively, together with the availability of monoclonal antibodies recognising epitopes dependent upon the integrity of the third extracellular loop of AE-1, have allowed us to study the organisation of the membrane domain of the mutant AE-1 found in South-east Asian ovalocytes (AE-1 SAG). The results suggest that the organisation of the whole membrane domain of AE-1 SAO is abnormal and that the organisation of other integral membrane proteins like those involved in expression of Rh blood group antigens may also be affected. Increased homo- and hetero-associations involving AE-1 SAO and other integral proteins may in turn result in reduced membrane flexibility. Purified protein 4.1 binds with 50-fold higher affinity to protein 4.1 depleted normal red cell membranes than to protein 4.1 depleted red cell membranes of Leach phenotype which lack Glycophorin C (GPC) and Glycophorin D (GPD). Experiments using purified protein 4.1 and p55 together with synthetic peptides corresponding to different regions of the cytoplasmic domain of Glycophorins C and D (GPC/D) demonstrate that protein 4.1 interacts directly with GPC through residues 82-98. They also show that p55 binds to GPC through residues 112-128. Since p55 also binds directly to protein 4.1 it is clear that protein 4.1 can bind to GPC through two different sites either directly through residues 82-98 or indirectly through p55. These results show that GPC and GPD provide major attachment sites for the red cell skeleton via protein 4.1 and that p55 is part of this complex.