Molecular basis of spectrin deficiency in beta spectrin Durham. A deletion within beta spectrin adjacent to the ankyrin-binding site precludes spectrin attachment to the membrane in hereditary spherocytosis.
We describe a spectrin variant characterized by a truncated beta chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderate hemolytic anemia with striking spherocytosis and mild spiculation of the red cells. We describe the biochemical characteristics of this truncated protein which constitutes only 10% of the total beta spectrin present on the membrane, resulting in spectrin deficiency. Analysis of reticulocyte cDNA revealed the deletion of exons 22 and 23. We show, using Southern blot analysis, that this truncation results from a 4.6-kb genomic deletion. To elucidate the basis for the decreased amount of the truncated protein on the membrane and the overall spectrin deficiency, we show that (a) the mutated gene is efficiently transcribed and its mRNA abundant in reticulocytes, (b) the mutant protein is normally synthesized in erythroid progenitor cells, (c) the stability of the mutant protein in the cytoplasm of erythroblasts parallels that of the normal beta spectrin, and (d) the abnormal protein is inefficiently incorporated into the membrane of erythroblasts. We conclude that the truncation within the beta spectrin leads to inefficient incorporation of the mutant protein into the skeleton despite its normal synthesis and stability. We postulate that this misincorporation results from conformational changes of the beta spectrin subunit affecting the binding of the abnormal heterodimer to ankyrin, and we provide evidence based on binding assays of recombinant synthetic peptides to inside-out-vesicles to support this model.
Hassoun
H
H
Department of Biomedical Research and the Division of Hematology/Oncology, St. Elizabeth's Hospital, Boston, Massachusetts 02135, USA.
Vassiliadis
J N
JN
Murray
J
J
Yi
S J
SJ
Hanspal
M
M
Ware
R E
RE
Winter
S S
SS
Chiou
S S
SS
Palek
J
J
D017487:Ankyrins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D002675:Child, Preschool; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004900:Erythroblasts / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D005260:Female; D014644:Genetic Variation; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000097:blood / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012156:Reticulocytes / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics*; D013103:Spherocytosis, Hereditary / Q000097:blood / Q000235:genetics*
D017487:Ankyrins; D017931:DNA Primers; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; C098729:spectrin Durham; D013049:Spectrin