Defective anion transport and marked spherocytosis with membrane instability caused by hereditary total deficiency of red cell band 3 in cattle due to a nonsense mutation.

Abstract

Cattle that showed moderate uncompensated anaemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth, were studied. SDS-PAGE, immunoblotting and electron microscope analysis by the freeze fracture method, showed that the erythrocytes of the proband lacked band 3 protein (anion exchanger 1, a transmembrane glycoprotein mediating Cl^-/bicarbonate ion exchange) completely. Sequence analysis of band 3 cDNA and genomic DNA from the proband showed a C→T substitution resulting in a nonsense mutation (CGA→TGA; Arg→Stop) at the position corresponding to codon 646 in human erythrocyte band 3 cDNA. The erythrocytes of the proband were deficient in spectrin, ankyrin, actin and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Thus, the erythrocyte membranes of the proband were extremely unstable and showed a reduced surface area in several ways, such as invagination, vesiculation and extrusion of microvesicles, leading to the formation of spherocytes. Total deficiency of band 3 also resulted in defective Cl^-/bicarbonate ion exchange, causing mild acidosis with decreases in the bicarbonate concentration and total CO₂ in the blood. The results show that band 3 contributes to red cell membrane stability, CO₂ transport, and acid-base homeostasis, but is not always essential for the survival of cattle.