A deletional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin Tokyo (beta 220/216).

A novel spectrin variant carrying a truncated beta-chain and designated Spectrin Tokyo (beta 220/216) is presented. It was associated with elliptocytosis and moderate uncompensated hemolysis. The dimer self-association was reduced. An increase of the alpha I 74-Kd fragment was detected upon partial trypsin digestion. Analysis of cDNA and genomic DNA showed a 1-base deletion in codon 2059 (GCC AGC-->GCA GCT; Ala-Ser-->Ala-Ala) that belongs to exon X of spectrin beta-gene. A missense sequence extended down to (new) codon 2075. Serine 2060, a potential phosphorylation site, was replaced by alanine. The shortened beta-chain failed to undergo phosphorylation in vitro. Spectrin Tokyo shared the same stop codon, overlapping normal codons 2076 and 2077 (CTG AAA), as Spectrin Nice (beta 220/216), which is caused by a dinucleotide insertion in codon 2046 and contains 2076 amino acids. However, for some reason, Spectrin Tokyo had a lower incorporation level into the membrane than Spectrin Nice.     

Elliptocytosis-associated spectrin Rouen (beta 220/218) has a truncated but still phosphorylatable beta chain.

Spectrin Rouen (beta 220/218) is a novel variant, carrying a shortened beta chain with an apparent molecular weight of 218 kDa. It was detected in a French family. All affected members suffered from haemolytic hereditary elliptocytosis. As other shortened beta chain variants described before, the beta Rouen chain is truncated at its carboxyl terminus. Spectrin Rouen is associated with a defect in spectrin dimer self-association and with an abnormally high amount of the alpha I 74 kDa peptide following partial tryptic digestion. Dimer reconstitution experiments from normal and abnormal purified Sp subunits indicated that the increased alpha I 74 kDa fragment is induced by the altered beta chain. However, spectrin Rouen is different from other mutants with a truncated beta chain in several respects: its amount is low (less than 10%) and the spectrin dimer self-associated defect is mild. Critically, the beta Rouen chain has retained the ability of undergoing phosphorylation, even though it is modified in its C-terminal region. These results, compared to those obtained with beta 220/214 spectrin Le Puy and beta 220/216 spectrin Nice, allowed better localization of the beta chain sites that can be phosphorylated by a membrane-bound casein kinase.     

An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216)

Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C-terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.     

Spectrin Oran (alpha II/21), a new spectrin variant concerning the alpha II domain and causing severe elliptocytosis in the homozygous state

We report on spectrin Oran (alpha II/21), a new spectrin variant found in an Algerian family. It was characterized by the absence of the spots that classically correspond to the alpha II domain using two-dimensional analysis of spectrin limit digests. On the contrary, the abnormal domain was represented by a new set of spots in the 21-Kd and 16-Kd regions, as demonstrated by Western blots using anti-alpha II domain polyclonal antibodies. Spectrin Oran (alpha II/21) was found in the homozygous state in two children belonging to two separate branches of the family. It yields a severe elliptocytosis. Spectrin self-association was altered. The variant was much more difficult to prove in the heterozygous state, in which it results in no clinical and virtually no morphological symptom. In all four parents involved, however, electrophoretic analysis and Western blots showed the existence of the alpha II 21-Kd and 16-Kd peptides. In one parent, who combines spectrin Oran (alpha II/21) and the alpha II type-2 polymorphism, the two-dimensional spots (52, 39, 34, and 29 Kd) were quantified and appeared reduced by 30%: there was an intermediary decrease of spectrin self-association in this person. In the three other parents, spectrin Oran combined with the alpha II type-1 polymorphism. The alpha II type-1 spots (46, 35, 30, and 25 Kd) appeared in normal range, and spectrin self-association was normal. Along with previous observations, the present data emphasize the large fluctuations of the alpha-variant percentage. Provided spectrin Oran was present in a sufficient proportion, we found an associated alteration of the beta II domain (that faces the alpha II domain in the spectrin dimer): the beta II 65-Kd fragment was reduced and the beta II 52-Kd fragment was reciprocally increased.     

A case of elliptocytosis associated with a truncated spectrin chain

A case of haemolytic anaemia with elliptocytosis is described, in which a large part of the smaller (beta) subunit of the spectrin is truncated, and has an apparent molecular weight of about 214 000 compared with about 230 000 for the normal chain. It is shown that this is not a product of adventitious proteolysis during lysis or extraction. At the same time about 35% of the total spectrin in the cells is liberated from the membrane as the dimer (which is present in normal cells to the extent of less than 10%). The truncated (beta') chain appears exclusively in this dimer fraction. The beta'-chain is incapable of phosphorylation by the endogenous cAMP-independent membrane kinase, and it may be inferred that the deleted segment of the chain contains both the spectrin self-association site and the residues normally phosphorylated. The alpha beta'-dimer is active with respect to participation in a ternary complex with its partnering proteins in the membrane cytoskeleton, F-actin and 4.1, confirming that the phosphorylation sites are not involved in the primary interaction with the other cytoskeletal proteins at the network junctions. The spectrin alpha-chain generates the terminal tryptic fragment of molecular weight 80 000 characteristic of normal spectrin, rather than the 74 000 molecular weight peptide derived from the alpha-chain in cases of hereditary elliptocytosis and pyropoikilocytosis, associated with anomalous self-association of spectrin dimer. Membrane cytoskeletons, extracted from the patient's red cells, undergo normal gelation on incubation with cAMP-independent kinase and ATP, and thus do not resemble those derived from hereditary spherocytosis cells. The properties of the anomalous spectrin resemble in most respects that described in a French family by Dhermy et al (1982).     

Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or beta-chain of spectrin through in vitro dimer reconstitution

Partial digestion of spectrin dimers in vitro has allowed the definition of domains. For example, the portions of the dimers that are involved in spectrin self-association are represented by the alpha I and the beta I domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd fragment and, more substantially, into a 74 Kd fragment. The intensity of the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the 74:alpha I ratio, is variable depending on the experimental conditions, eg, in fine, on the conformation of the alpha I domain. A number of cases of hereditary elliptocytosis (HE) are associated with an increase of the 74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality. Several lines of evidence have suggested that the causal mutations may lie in the alpha- or the beta-chain, a point of importance before one undertakes studies at the gene level. In order to address this question, we reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of various origins, and then carried out partial digestion and assayed the Sp alpha I/74 abnormality. The patterns obtained with reconstituted dimers were nearly identical to those of native dimers. We applied the assay to three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we previously designated spectrin Nice and whose beta-chain lacks a 4 Kd fragment in its C-terminal region; and two distinct variants that we found in two unrelated white families and that we provisionally designated spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in all kinds of dimers that harbored the beta-chain of spectrin Nice, or the alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we confirmed that spectrin Nice is a (alpha I/74) beta-variant, and established that both spectrin Lyon and spectrin Culoz are (alpha I/74) alpha-variants. The present assay may be extended to any spectrin variant displaying the Sp alpha I/74 abnormality.     

Occurrence of the αI 22 Arg→His (CGT→CAT) spectrin mutation in Tunisia: potential association with severe elliptopoikilocytosis

A category of spectrin alpha I domain variants are manifested by the increase of the alpha I 74 kDa fragment at the expense of the parent 80 kDa fragment following partial tryptic digestion. We describe a particular case of alpha I/74 abnormality in a Tunisian family. The propositus was severely ill and had an elliptopoikilocytosis. To the contrary, his father, who carried the same alpha I/74 variant, displayed no clinical signs and a few elliptocytes. The increase of the alpha I 74 kDa fragment was more pronounced in the propositus than in his father. Unexpectedly, the spectrin content was reduced to similar (and limited) extents in both of them, and the father displayed nearly as pronounced an increase of the spectrin dimer percentage as the propositus following low ionic strength extraction. In vitro spectrin dimer reconstitution experiments indicated that the primary mutation was located in the alpha-chain itself (not in the beta-chain as is the case in some alpha I/74 mutants). Following polymerase chain reaction (PCR) amplification, cloning and sequencing of exon 2 of spectrin alpha-gene in the father, we found the G----A substitution at position 2 of codon 22 (CGT----CAT; Arg----His). This mutation has been recently discovered in a family of French descent. Dot blot hybridization confirmed that the substitution was transmitted with the alpha I/74 abnormality. As previously shown, the enhancement of its expression level in the propositus, with respect to the father, was accounted for by the presence of a factor carried by the alpha-spectrin allele in trans and transmitted by the mother.     

Abnormal tryptic peptide from the spectrin α-chain resulting from α- or β-chain mutations: two genetically distinct forms of the Sp αI/74 variant

Limited tryptic digestion of native spectrin (Sp) has revealed several variants in hereditary pyropoikilocytosis (HPP) and in a subset of patients with hereditary elliptocytosis (HE). In most cases, tryptic peptide corresponding to the alpha I (N-terminal) 80 kD domain is wholly or partially replaced by smaller fragments. These variants are provisionally designated according to the molecular weight of the most prominent new peptide. Partial amino acid sequences of the abnormal peptides and DNA analysis of the alpha-spectrin gene have shown that most variants result from substitution or insertion of an amino acid in the alpha I-domain. However, similar investigations did not detect any such abnormality in the spectrin alpha I-domain of an HE black kindred with one of the spectrin variants called Sp alpha I/74. In this kindred, restriction fragment length polymorphism studies and transmission of the genetic polymorphism relative to the alpha II-domain excluded the involvement of the alpha-chain in the pathological process. To ascertain whether the abnormal alpha I 74 kD peptide might be caused by a beta-chain mutation, we reconstituted hybrid dimers combining normal and HE Sp-chains. The tryptic peptide patterns of spectrin hybrid dimers containing HE alpha-chain and control beta-chain showed a normal 80 kD tryptic product. In contrast, the hybrid dimer containing normal alpha-chain and HE beta-chain gave rise to increased 74 kD peptide at the expense of the 80 kD, demonstrating that the mutation in this family resides in the beta-chain. The same method was used to show that in two other unrelated white kindreds, the elevated 74 kD peptide arose from a Sp alpha-chain defect. Thus an alteration in tryptic susceptibility within the N-terminal domain of the spectrin alpha-chain can be directed by a mutation in the beta-chain. The hybridization technique affords a definitive means of distinguishing between alpha- and beta-chain mutants.     

Defective spectrin dimer-dimer association with hereditary elliptocytosis.

We examined erythrocytes from 18 patients with hereditary elliptocytosis. Spectrin from eight patients (referred to as type 1) was defective in dimer-dimer association as demonstrated in two ways. First, there was an increased amount of spectrin dimer with a concomitant decrease in tetramer as measured in erythrocyte membrane preparations extracted at 0 degrees C under low-salt conditions (the amount of spectrin dimer was 15-33% of total spectrin species compared with a normal range of 3-7%). Second, the equilibrium constants of spectrin dimer-dimer association were decreased in both solution and in situ membrane. Spectrin from the remaining 10 patients (referred to as type 2) showed normal dimer-dimer association. Membrane skeletons, produced from ghosts of both types of hereditary elliptocytosis by Triton X-100 extraction, were unstable when mechanically shaken. Because spectrin tetramers, but not dimers, can crosslink actin, we postulate that the defective spectrin dimer-dimer association in type 1 diminishes actin crosslinking and thus is responsible for membrane skeletal instability. A defective protein-protein association in type 2, however, remains to be identified.     

Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain

Spectrin Tunis (Sp alpha I/78) is an alpha l domain variant that causes asymptomatic elliptocytosis in the heterozygote state. It is manifested by a reduction of spectrin dimer self-association and by the development of a major 78-Kd fragment at the expense of the alpha l 80- Kd fragment upon spectrin-limited digestion. Amino acid sequence analysis, following peptide transfer onto Immobilon membranes, showed that the 78-Kd fragment results from a sensitized cleavage after lysyl residue 10. Using a 13.5-kb genomic alpha-spectrin probe and the Xbal, Pvull, and Mspl polymorphic sites detected with this probe, we concluded that spectrin Tunis is associated with the + - + haplotype (in the above order). Twenty mer oligonucleotides, complementary to genomic segments from introns 2 and 3, respectively, were synthesized. We then performed DNA amplification and sequencing. In the two investigated carriers of spectrin Tunis, we found the C----T base substitution of the codon corresponding to position 35 of the alpha l domain (CGG----TGG; Arg----Trp). The mutation lies in the last part of an alpha helix that extends from residues 9 to 44 of partial repeat alpha 1' and is comparable with helix 3 of full repeats 1 to 5. The modified proteolytic site, located 25 amino acid residues upstream, occurs at the beginning of the helix.     

Sp alpha I/65 hereditary elliptocytosis in North Africa

The Sp alpha I/65 variant of the spectrin has been recently described in black people with hereditary elliptocytosis (HE). The present study reports on a similar Sp alpha I/65 variant in nine North African persons belonging to four unrelated families. The abnormality was associated with a variable degree of elliptocytosis. In one case, red cell morphology was normal. In the nine carriers of the biochemical abnormality, the spectrin dimer self-association was defective. The association constant was reduced: 0.65 to 1.7 X 10(5) M-1 (controls: 4.6 +/- 0.5 X 10(5) mM-1 (n = 21)); in six cases, there was a higher level of spectrin dimer in the low ionic strength extract at 4 degrees C: 13.0 to 19.7% (controls: 6.4 +/- 2.1% (n = 7)). Limited tryptic digests of spectrin from the nine persons revealed a decrease of the 80,000-dalton alpha-1 domain, and the concomitant appearance of a peptide with a molecular weight of 65,000 daltons and an isoelectric point ranging from 5.0 to 5.1. There was a correlation between the proportion of the 65,000-dalton fragments, the defect of spectrin self-association, and the extent of morphological alteration. This is the first large series concerning a spectrin abnormality in non-black persons. In North Africa, cases of HE that are not due to a protein 4.1 defect have turned out so far to be associated with the Sp alpha I/65 variant.     

A new abnormal variant of spectrin in black patients with hereditary elliptocytosis

Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.4 and 2.4 X 10(5) M-1 for a normal value of 6 +/- 0.4 X 10(5) M-1. Spectrin (Sp) from HE patients and normal volunteers (32 black and 22 white subjects) was submitted to limited tryptic digestion, followed by one- or two-dimensional separation of the peptides. Peptide patterns of crude Sp from all seven HE patients exhibited a marked and reproducible decrease in 80,000-dalton peptide (previously identified as the dimer- dimer interaction domain of the alpha-chain) and a concomitant appearance of a novel 65,000-dalton peptide. A minor fragment at 28,000 daltons was also decreased. Tryptic digestion of HE spectrin dimer and tetramer (SpT), isolated after the SpD self-association procedure in solution, revealed modifications (decrease in the 80,000-dalton peptide and presence of a 65,000-dalton peptide) predominantly in HE SpD when peptide patterns of HE SpT were quite similar to control SpT patterns. Immunoblots with anti-alpha-chain antibodies revealed that the 65,000- dalton peptide derived from the alpha-chain. Kinetic studies of Sp digestion showed that the 65,000-dalton peptide did not result from further digestion of a 74,000 intermediate and was not a precursor of 46,000- to 50,000-dalton peptides. These results show a new structural defect of Sp-alpha-chain, associated with a defective Sp dimer self- association in HE.     

Family of hereditary elliptocytosis with abnormalities of spectrin function (Sp alpha 1/74)

We have experienced a case of cytohemolytic hereditary elliptocytosis (HE) in a six-year-old boy. Metabolisms of the erythrocytic membrane were investigated on the members from his pedigree. The results were as follows; 1) The presence or absence of ovalocytic HE were studied in his pedigree. 2) Failure in the process of spectrin dimer to tetramer conversion was found. 3) Although abnormality existed in conversion of D to T by the patient's alpha-chain spectrin and normal beta-chain spectrin, no abnormality was recognized when normal alpha-chain and the patient's beta-chain were combined. 4) Decrease of the alpha-chain (80 kd) domain and appearance of abnormal (74 kd) spot were found by two dimensional peptide mapping of spectrin. 5) In his pedigree, neither patients with hereditary pyropoikilocytosis (HPP) nor carrier states were recognized. In summary, this patient's pedigree was considered to be HE [SP alpha 1/74]. This case appears to be the first case in Japan and only few cases have been reported in the world literature.     

Poikilocytic hereditary elliptocytosis associated with spectrin Alexandria: an alpha I/50b Kd variant that is caused by a single amino acid deletion

Hereditary elliptocytosis (HE) is a heterogeneous disorder of red blood cells frequently associated with abnormal limited tryptic digestion of the alpha I domain of spectrin and impaired spectrin dimer self- association. We studied two related individuals with poikilocytic hereditary elliptocytosis (HE) of different severity. Limited tryptic digestion of spectrin from these individuals showed the presence of a variant alpha I/50b Kd peptide at the expense of the normal alpha I/80 Kd peptide. Amino acid sequence analysis of the abnormal peptide showed that the proteolytic cleavage occurred after the arginine at position 470 of the alpha spectrin chain. Spectrin from these patients had an impaired ability to undergo self-association, as evidenced by increased amounts of spectrin dimers in 4 degrees C extracts of erythrocyte membrane from affected individuals. The polymerase chain reaction was used to study the DNA sequence of the alpha spectrin gene encoding the region of the alpha spectrin chain surrounding the abnormal proteolytic cleavage site. We detected the in-frame deletion of the trinucleotide CAT, encoding histidine 469, two amino acid residues to the N-terminal side of the abnormal proteolytic cleavage site between residues 470 and 471. Similar to many other defects of spectrin associated with HE, this deletion occurs in helix three of repeat 5 of the proposed triple helical model of spectrin repeats.     

Identification of functional domains of human erythrocyte spectrin.

Isolated human erythrocyte spectrin is a dimer of two unique polypeptide chains. The dimer (alpha beta) undergoes reversible salt- and temperature-dependent association to form (alpha beta)2 tetramers. Spectrin also binds with high affinity to a protein receptor on the cytoplasmic surface of erythrocyte membrane vesicles. By cleavage of spectrin at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid, a 50,000-dalton peptide fragment has been isolated which inhibits the binding of spectrin to erythrocyte membrane vesicles. This peptide arises from a terminal region of the beta chain. An 80,000-dalton peptide generated by restricted trypsin digestion binds preferentially to dimeric spectrin. This peptide arises from a terminal portion of the alpha chain. Multiple peptides involved in noncovalent associations between the chains have also been identified. These associations indicate that the two subunits of spectrin are aligned parallel to one another and that the tetramer formation site and the high-affinity membrane binding site are in close proximity to one another.     

A large erythroid spectrin β-chain variant

A large variant of erythrocyte beta-spectrin was found in a child presenting with hereditary elliptocytosis and anaemia. This polypeptide was phosphorylated, cross-reacted with normal beta-spectrin in immunoblotting and formed a dimer with alpha-spectrin that co-purified with normal alpha beta dimer. The molecular weight was estimated to be 330 kD by SDS gel electrophoresis, which is 84 kD (35%) larger than the normal beta-chain. This variant has been tentatively named spectrin Detroit (beta Detroit). Tryptic digests demonstrated a coexisting alpha-spectrin variant Sp alpha I/65 in the propositus, his father and a paternal uncle. Anaemia and elliptocytosis was associated with Sp alpha I/65 rather than beta Detroit, since other family members with beta Detroit in whom alpha-spectrin was normal had no morphological or clinical abnormalities. Family members were identified who had normal alpha-spectrin but were heterozygotic for the large beta-spectrin. Their erythrocyte membranes were more rigid and fragile than normal. The fragility is probably a consequence of both weaker dimer association and spectrin deficiency. Variant spectrin dimers (alpha beta Detroit) had a reduced self-association constants. Binding to ankyrin was normal. Instability of beta Detroit during erythropoiesis is suggested by the fact that it comprises only 25% of the beta-spectrin in beta Detroit heterozygote erythrocytes, and total spectrin was reduced by 20%. Although beta Detroit has some functional defects, this 84 kDa insert in erythrocyte spectrin is compatible with nearly normal function.     

Ultrastructural studies of the interaction of spectrin with phosphatidylserine liposomes

Spectrin was shown previously to interact with phosphatidylserine and phosphatidylethanolamine, which are preferentially localized in the inner half of the membrane lipid bilayer, but this interaction is not well characterized. In the present study we used electron microscopy of rotary-shadowed platinum replicas of spectrin dimer-phosphatidylserine complexes to study the interaction of spectrin with phosphatidylserine vesicles. At a spectrin concentration of 0.6 mg/mL, 60% of spectrin dimers were associated with phosphatidylserine vesicles and at a spectrin concentration of 1.2 mg/mL, some vesicles were crosslinked by spectrin dimers. The length of the protruding segment of spectrin dimer from the liposome edge ranged from 400 to 960A degrees and the contact region to phosphatidylserine extended 272 +/- 144A degrees from either end of the molecule. Therefore, these data are consistent with multiple binding sites to phosphatidylserine throughout the spectrin dimer molecule. Spectrin tetramers, when bound to phosphatidylserine liposomes, extended 1804 +/- 79A degrees from the liposome edge and crosslinked liposomes, suggesting that some of the binding sites to phosphatidylserine vesicles is in the proximity of the tail end of spectrin. The association between spectrin dimers to phosphatidylserine was demonstrated by nondenaturing gel electrophoresis. The complexes were separated into multiple bands with molecular weight of 1.4 X 10(6), 1.8 X 10(6), and 2.3 X 10(6). These bands did not represent self- associated spectrin oligomers, since postincubation treatment with Triton-X-100 dissociated them into spectrin dimers. Furthermore, these spectrin high molecular weight bands, as visualized by Coomassie blue absorbance, closely corresponded to the 14C-phosphatidylserine distribution. These data provide ultrastructural and biochemical evidence that spectrin binds to phosphatidylserine at multiple sites including the tail end region.     

Mammalian alpha I-spectrin is a neofunctionalized polypeptide adapted to small highly deformable erythrocytes

Mammalian red blood cells, unlike those of other vertebrates, must withstand the rigors of circulation in the absence of new protein synthesis. Key to this is plasma membrane elasticity deriving from the protein spectrin, which forms a network on the cytoplasmic face. Spectrin is a tetramer (alphabeta)(2), made up of alphabeta dimers linked head to head. We show here that one component of erythrocyte spectrin, alphaI, is encoded by a gene unique to mammals. Phylogenetic analysis suggests that the other alpha-spectrin gene (alphaII) common to all vertebrates was duplicated after the emergence of amphibia, and that the resulting alphaI gene was preserved only in mammals. The activities of alphaI and alphaII spectrins differ in the context of the human red cell membrane. An alphaI-spectrin fragment containing the site of head-to-head interaction with the beta-chain binds more weakly than the corresponding alphaII fragment to this site. The latter competes so strongly with endogenous alphaI as to cause destabilization of membranes at 100-fold lower concentration than the alphaI fragment. The efficacies of alphaI/alphaII chimeras indicate that the partial structural repeat, which binds to the complementary beta-spectrin element, and the adjacent complete repeat together determine the strength of the dimer-dimer interaction on the membrane. Alignment of all available alpha-spectrin N-terminal sequences reveals three blocks of sequence unique to alphaI. Furthermore, human alphaII-spectrin is closer to fruitfly alpha-spectrin than to human alphaI-spectrin, consistent with adaptation of alphaI to new functions. We conclude that alphaI-spectrin represents a neofunctionalized spectrin adapted to the rapid make and break of tetramers.     

Spectrin Jendouba: an alpha II/31 spectrin variant that is associated with elliptocytosis and carries a mutation distant from the dimer self-association site.

Spectrin Jendouba (alpha II/31) was found in a Tunisian family. In the heterozygous state, it is associated with asymptomatic elliptocytosis and a minimal defect in spectrin dimer self-association. On partial digestion of spectrin with trypsin, an abnormal cleavage appeared following Lys 788. Peptide and DNA sequencing indicated that the responsible mutation is alpha 791 Asp----Glu (GAC----GAA). As in most alpha-spectrin variants associated with elliptocytosis, the change alters helix 3 of the proposed triple helical model of spectrin structure. Modified helix 3 in repeat alpha 8 is the most distant from the N-terminus of alpha-spectrin in known variants associated with elliptocytosis.     

Expression of spectrin in normal and malignant erythropoiesis

Spectrin is a major constituent of the erythrocyte membranoskeleton. The occurrence of spectrin during normal and malignant erythropoiesis was investigated by immunofluorescence using a monospecific rabbit anti-human spectrin antiserum. The expression of spectrin was correlated to the presence of glycophorin A, which is an early and specific marker for erythroid cells. The expression of spectrin during normal erythroid differentiation coincided with that of glycophorin A. Both markers were already present in the proerythroblasts. Spectrin was also found in leukaemic cells from patients with acute erythroleukaemia and erythroid blast crisis of chronic myelogenous leukaemia. In a large panel of human haematopoietic cell lines only those with erythroid phenotype (K 562 and HEL) stained positively for spectrin. It is concluded that spectrin appears early in the erythroid maturation. It is expressed both in normal and malignant erythroid precursors. Spectrin can be used as a marker for erythroid blasts in the diagnosis of erythroleukaemias.