Normoblastosis, a murine model for ankyrin-deficient hemolytic anemia, is caused by a hypomorphic mutation in the erythroid ankyrin gene Ank1

Abstract

Ankyrin deficiency is one of the most common causes of hereditary spherocytosis in humans. A spontaneous mutation, normoblastosis (Ank1nb), discovered in 1969 in a mouse stock maintained at the Jackson Laboratory, provides an important animal model for these human ankyrin-deficient anemias. Study of this model has led to the finding of multiple isoforms of Ank1 as well as Ank1nb-related pathology in nonerythroid tissues. To enhance the usefulness of this model, we have identified the Ank1nb mutation as the deletion of a guanosine residue in exon 36 of the erythroid ankyrin gene (Ank1). This results in a frame shift that introduces a stop 13 codons downstream and predicts a 157 kDa nb-ankyrin lacking the regulatory domain but including intact membrane- and spectrin-binding domains. By epitope scanning on immunoblots, we show that a previously reported protein (p150) found in nb reticulocytes is the predicted nb-ankyrin. Existing evidence indicates that this protein is functional, making the normoblastosis mutation a hypomorph rather than a null as originally thought. The nb-ankyrin provides an explanation for the milder phenotype displayed by nb/nb animals relative to the murine spectrin-deficient anemias, spherocytosis (Spna1(sph), Spna1(sph-1J), Spna1(sph-2BC), Spna1(sph-DEM)) and jaundiced (Spnb1(ja)), and suggests that truncated ankyrins could be useful in gene replacement therapy.

Human Rhesus-associated glycoprotein mediates facilitated transport of NH 3 into red blood cells

“…These mice have primary defects in Ank1 (nb͞nb) (44) or ␣-spectrin (sph͞sph) (45). RBCs from nb͞nb mice exhibit a sharp reduction of Rh and Rhag proteins but normal levels of CD47 (7), which is also true for RBCs from sph͞sph mice (Table 4).…”

Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant

“…The truncated ANK-1 protein in the nb/nb mouse lacks the regulatory domain, but includes the membrane and spectrin-binding domain. 20 The levels of the other critical membrane proteins are preserved in the nb/nb mice, with only spectrin levels being reduced to 50% of wild-type. The nb/nb mice display normal membrane skeletal ultrastructure, prompting the conclusion that ankyrin was not required for the formation of a stable 2-dimensional spectrin-based skeleton.…”

“…28 More recently, the nb mutation has been shown to be a hypomorphic allele of Ank-1, producing a truncated protein of 157 kDa. 20 The expression of this protein suggests that the functional consequences ascribed to Ank-1 deficiency in the nb/nb mice may well differ with analysis of mice carrying an Ank-1 null mutation.…”

“…4 Only a single mouse model of ankyrin deficiency has been described, the spontaneous normoblastosis mouse (nb/nb). 20 This line carries a deletion of a guanosine residue in exon 36 that leads to a frame shift that introduces a premature stop codon after the addition of 13 residues, resulting in the production of a 157-kDa protein. The wild-type ankyrin-1 (ANK-1) protein is 210 kDa and contains 3 major functional domains, an N-terminal 89-kDa membrane-binding domain, a 62-kDa spectrin-binding domain, and a C-terminal 55-kDa regulatory domain.…”

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

“…Thus, nb/nb (39) mice express Ͻ5% of the normal amount of wildtype ankyrin, but this loss is partially compensated for by the production of a mutant ankyrin that lacks a regulatory domain, but retains the band 3 and spectrin binding domains (48). Red cells from nb/nb mice retain relatively normal looking architecture by electron microscopy with an ankyrin immunoreactive protein detected in the normal position in their red cell membranes (49).…”