Interactions of spectrin in hereditary elliptocytes containing truncated spectrin β-chains

Abstract

An abnormal spectrin, in which one subunit is truncated, has been detected in a large German family. The inheritance is autosomal dominant. The affected members of the family suffer in widely varying degree from a microcytic hemolytic anemia. The red cell morphology varies correspondingly from smooth elliptocytes to predominantly poikilocytes. The abnormal spectrin makes up ~30% of the total and is almost entirely present as the dimer. The truncated chain is not phosphorylated by the endogenous cAMP-independent kinase, and it has been identified as a chain of β-type, using monoclonal antibodies. Because a univalent terminal spectrin α-chain fragment will bind to normal dimers with an association constant lower by only a factor of two than that for the self-association of the dimers, it would be expected that the mutant dimers (αβ') would readily enter into an association with normal (αβ) dimers to give α2ββ' tetramers (though not with each other). In dilute solution this is indeed observed, and the diminution in tetramer concentration when 30% of normal spectrin is replaced by αβ' dimers, amounts to only a small proportion. Moreover, in the membrane skeleton, if there is pairwise apposition of dimer units, only 9% of pairings will be between units that cannot associate. We have shown that the failure of αβ' dimers to enter into heterologous associations in situ is not due to the elimination of the ankyrin binding site near the truncated end of the β-chain: this site is fully functional, as judged by rebinding to spectrin-depleted vesicles. When the spectrin is extracted from the membrane in the cold, the material released initially consists almost entirely of αβ' dimers; when the spectrin of normal membranes is partly dissociated to dimers in situ by warming at low ionic strength, extraction in the cold then leads similarly to much more rapid release of the dimer than of the tetramer. The similar rates of liberation of normal and abnormal dimer make it unlikely that the interaction of the latter with the membrane is in any way defective. When mixtures of αβ and αβ' dimers are bound to spectrin-depleted inside-out membrane vesicles from normal cells and tetramers are allowed to form by equilibration at 30°C, the proportion of the abnormal species appearing in the tetramer is much lower than would be expected on a statistical basis. The relation of the self-association equilibrium on the membrane to that of spectrin in dilute solution is analyzed.