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

We studied bovine subjects that exhibited a moderate uncompensated ane mia with hereditary spherocytosis inherited in an autosomal incomplete ly dominant mode and retarded growth. Based on the results of SDS-PAGE , immunoblotting, and electron microscopic analysis by the freeze frac ture method, we show here that the proband red cells lacked the band 3 protein completely. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C --> T substitution resulting in a nonsense muta tion (CGA --> TGA; Arg --> Stop) at the position corresponding to codo n 646 in human red cell band 3 cDNA. The proband red cells were defici ent in spectrin, ankyrin, actin, and protein 4.2, resulting in a disto rted and disrupted membrane skeletal network with decreased density. T herefore, the proband red cell membranes were extremely unstable and s howed the loss of surface area in several distinct ways such as invagi nation, vesiculation, and extrusion of microvesicles, leading to the f ormation of spherocytes. Total deficiency of band 3 also resulted in d efective Cl-/HCO3- exchange, causing mild acidosis with decreases in t he HCO3- concentration and total CO2 in the proband blood. Our results demonstrate that band 3 indeed contributes to red cell membrane stabi lity, CO2 transport, and acid-base homeostasis, but is not always esse ntial to the survival of this mammal.