Deficiency of protein 4.2 in erythrocytes from a patient with a Coombs negative hemolytic anemia. Evidence for a role of protein 4.2 in stabilizing ankyrin on the membrane.

A patient with a mild hemolytic anemia and osmotically fragile, spherocytic erythrocytes was studied. Analysis of the erythrocyte membrane proteins by SDS-PAGE revealed a deficiency of protein 4.2 (< 0.10% of normal). The protein 4.2- deficient erythrocytes contained normal amounts of all other membrane proteins, although the amount of band 3 was slightly reduced and the amount of band 6 (G3PD) was slightly elevated. The spectrin content of these cells was normal, as measured by both SDS-PAGE and radioimmunoassay. Erythrocytes from the patient's biologic parents were hematologically normal and contained normal amounts of protein 4.2. Im- munological analysis using affinity purified antibodies revealed that the patient's protein 4.2 was composed of equal amounts of a 74-kD and 72-kD protein doublet, whereas the normal protein was composed primarily of a 72-kD monomer. Proteo- lytic digestion studies using trypsin, a-chymotrypsin and pa- pain demonstrated that the patient's protein 4.2 was similar but not identical to the normal protein. Binding studies showed that the protein 4.2-deficient membranes bound purified pro- tein 4.2 to the same extent as normal membranes, suggesting that the membrane binding site(s) for the protein were normal. Depleting the protein 4.2-deficient membranes of spectrin and actin resulted in a loss of nearly two-thirds of the membrane ankyrin, whereas similar depletion of normal membranes re- sulted in no loss of ankyrin. Repletion of the protein 4.2-defi- cient membranes with purified protein 4.2 before spectrin-actin extraction partially prevented the loss of ankyrin. These re- sults suggest that protein 4.2 may function to stabilize ankyrin on the erythrocyte membrane. Introduction A major determinant of erythrocyte membrane shape and sta- bility is the membrane cytoskeleton, a two-dimensional pro- tein lattice located on the inner surface of the cell membrane. It is composed of two major supporting proteins, spectrin and actin; two linking proteins, ankyrin and protein 4.1; and their respective attachment sites on the cytoplasmic poles of band 3 and glycophorin (1). Defects or deficiencies in the protein