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Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 92 No. 7 (October 1), 1998: pp. 2591-2593 CORRESPONDENCE Reduced Spectrin-Ankyrin Binding in a South African Hereditary Elliptocytosis Kindred Homozygous for Spectrin St Claude     LETTER To the Editor: Attached to and supporting the inner leaflet of the erythrocyte membrane is a two-dimensional network of spectrin filaments that crosslink actin. Spectrin heterodimers consist of an  and  monomer closely associated in an antiparallel fashion. Spectrin is divided into five  (I-V) and four  (I-IV) spectrin structural domains by tryptic digestion.1 The primary structures of both chains are dominated by tandemly repeated 106-amino acid homologous repeat motifs2 that fold into triple helical bundles. Spectrin attaches to the lipid bilayer through an association with the integral band 3 protein via ankyrin, which binds repeats 15 and 16 of  spectrin. Spectrin dimers self-associate into tetramers in a head-to-head fashion via reciprocal interactions of the spectrin repeat with repeat 17 of  spectrin.1 Hereditary elliptocytosis (HE) is a disorder characterized by elliptocytes on peripheral blood smears and is most commonly caused by a spectrin dimer self-association defect.3 Two probands from a white South African kindred with severe HE, characterized by partial spectrin deficiency in the membrane, 25% spectrin dimers,4 and severely decreased spectrin-ankyrin binding,5 were further investigated to identify the underlying spectrin mutation. The close association of spectrin subunits in the heterodimer allows a defect in one chain to manifest itself as an alteration observed in the second chain. To identify the defective proband spectrin subunit, reconstituted hybrid spectrin dimers prepared from control (C) and proband (P) monomers6 were assayed. The hybrid spectrin-ankyrin binding assays in Fig 1 show the effect of increasing amounts of hybrid spectrin dimer competitor on the amount of control 125I-labeled spectrin dimers bound to spectrin depleted inside out vesicles. CC and CP were better able to compete with the labeled control spectrin for free ankyrin binding sites than PC and PP. Thus, a proband  spectrin defect reduces the ankyrin binding of the adjacent  spectrin. Quantitation of hybrid spectrin dimer self-association by densitometric scanning of nondenaturing gels indicated that the proband  spectrin also reduced dimer self-association, whereas proband  spectrin had no effect. View larger version (22K): [in this window] [in a new window]   Fig 1. Competitive hybrid spectrin-ankyrin binding assays. Hybrid spectrins, formed from control (C and C) and proband (P and P) spectrin monomers, were bound to spectrin-depleted inside-out vesicles (IOV) in the presence of a constant amount of 125I-labeled control spectrin dimer. The amount of bound control spectrin was plotted versus the amount of hybrid spectrin competitor added. All binding data are shown as the mean of duplicates that had ranges less than + or 8.5%. Proband  spectrin reduced the ankyrin binding of hybrid spectrins. Structural analysis of proband spectrin using tryptic digestion showed that the II domain was altered: on peptide maps there was an acidic shift of the pI of the 46-kD peptide and the 35-kD and 30-kD peptides were absent. These data indicated a defect between amino acids 916-981. Reticulocyte mRNA and genomic DNA analysis indicated that the probands were homozygous for a T G transversion 13 bp from the  spectrin intron 19/exon 20 boundary named Spectrin Johannesburg7,8 or Spectrin St Claude.9 This mutation creates a 3 acceptor splice site resulting in the expression of equal quantities of two abnormal messages. One message contains an in-frame 12-bp intron 19 insertion that introduces a translation stop codon and produces a truncated protein not incorporated into the membrane. In the second message exon 20 is excised by the spliceosome due to recognition of the stop codon. This mutant  spectrin lacks amino acids 935-965, which delete the B helix of the 9 repeat within the II domain. This mutation perturbs the conformation of the spectrin heterodimer, which reduces dimer self-association and impairs the binding of  spectrin to ankyrin via long-range interactions. The ankyrin binding and dimer-dimer contact sites of  spectrin are in contiguous repeats 15-17 and, therefore, a single disruptive influence could affect both functions. Approximate models of the relative positions of the spectrin triple helical bundles of each monomer in the heterodimer place repeat 9 opposite either 1210 or 14.11 This is in close proximity to repeats 15-17. The altered 9 conformation may thus disrupt ankyrin binding and dimer self-association by transmission of a steric effect along  spectrin and subsequently to 15-17. The disruptive effect of the mutant repeat 9 may also be propagated further to the N-terminal repeat and, hence, influence the  spectrin dimer self-association site. The kindred is of Afrikaans origin and the parents are apparently unrelated. Because the probands are homozygotes, both parents are obligate heterozygotes. The prevalence of the Spectrin St Claude allele was investigated in unrelated white South African individuals. Two mutant alleles out of 134 were detected, which contrasts with white subjects of French origin where the allele was not detected.9 The partial spectrin deficiency in the probands' erythrocyte membranes, which is a result of the spectrin-ankyrin binding defect, destabilizes the lipid bilayer and causes spherocytes. The reduced membrane spectrin content in concert with the mild dimer self-association defect further weakens the membrane skeleton and allows deformation of the erythrocytes into elliptocytes and poikilocytes. Our studies illustrate how a single point mutation in the  spectrin gene impairs functions of both the  and  spectrin proteins, resulting in qualitative and quantitative membrane abnormalities. These have profound effects on red blood cell morphology and survival, manifesting as severe hemolytic anemia. Jonathan P.W.G. Burke Deon Van Zyl Stan S. Zail Theresa L. Coetzer Department of Haematology South African Institute for Medical Research and the University of the Witwatersrand Johannesburg, South Africa        REFERENCES 1. Winkelmann JC, Forget BG: Erythroid and non-erythroid spectrins. Blood 81:3173, 1993[Abstract/Free Full Text] 2. Speicher DW, Marchesi VT: Erythrocyte spectrin is comprised of many homologous triple helical segments. Nature 311:177, 1984[Medline] [Order article via Infotrieve] 3. Palek J, Jarolim P: Hereditary spherocytosis, elliptocytosis, and related disorders, in Beutler E, Litchman MA, Coller BS, Kipps TJ (eds): Williams Hematology (ed 5). New York, NY, McGraw Hill, 1995, p 536 4. Coetzer TL, Zail SS: Spectrin tetramer-dimer equilibrium in hereditary elliptocytosis. Blood 59:900, 1982[Abstract/Free Full Text] 5. Zail SS, Coetzer TL: Defective binding of spectrin to ankyrin in a kindred with recessively inherited hereditary elliptocytosis. J Clin Invest 74:753, 1984 6. LeComte MC, Feo C, Gautero H, Bournier O, Galand C, Garbarz M, Boivin P, Dhermy D: Severe recessive poikilocytic anaemia with a new spectrin alpha-chain variant. Br J Haematol 74:497, 1990[Medline] [Order article via Infotrieve] 7. (abstr, suppl 1) Burke J, Zail SS, Coetzer TL: Spectrin Johannesburg: An abnormal spectrin II domain due to partial exon skipping decreases spectrin self-association and ankyrin binding resulting in hereditary elliptocytosis. Blood 90:4a, 1997 8. Burke JP, Zail SS, Coetzer TL: Exon skipping caused by a stop codon. Proceedings of the South African Society of Biochemistry and Molecular Biology, 14th Conference, Grahamstown, South Africa, January 1997, p 46 (abstr) 9. Fournier CM, Nicolas G, Gallagher PG, Dhermy D, Grandchamp B, LeComte MC: Spectrin St Claude, a splicing mutation of the human alpha-spectrin gene associated with severe poikilocytic anemia. Blood 89:4584, 1997[Abstract/Free Full Text] 10. DeSilva TM, Harper SL, Kotula L, Hensley P, Curtis PJ, Otvos L Jr, Speicher DW: Physical properties of a single-motif erythrocyte spectrin peptide: A highly stable independently folding unit. Biochemistry 36:3991, 1997[Medline] [Order article via Infotrieve] 11. Speicher DW, Weglarz L, DeSilva TM: Properties of human red cell spectrin heterodimer (side-to-side) assembly and identification of an essential nucleation site. J Biol Chem 267:14775, 1992[Abstract/Free Full Text] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Burke, J. P.W.G. Articles by Coetzer, T. L. Search for Related Content PubMed PubMed Citation Articles by Burke, J. P.W.G. Articles by Coetzer, T. L. Social Bookmarking                   What's this?

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