Spectrin-based skeleton as an actor in cell signaling.

This review focuses on the recent advances in functions of spectrins in non-erythroid cells. We discuss new data concerning the commonly known role of the spectrin-based skeleton in control of membrane organization, stability and shape, and tethering protein mosaics to the cellular motors and to all major filament systems. Particular effort has been undertaken to highlight recent advances linking spectrin to cell signaling phenomena and its participation in signal transduction pathways in many cell types.

Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.

A common type of the spectrin alpha I 46-50a-kD peptide abnormality in hereditary elliptocytosis and pyropoikilocytosis is associated with a mutation distant from the proteolytic cleavage site. Evidence for the functional importance of the triple helical model of spectrin.

We studied nine individuals from five unrelated families with alpha I/46-50a hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP), including one of the original HHP probands first reported by Zarkowsky and colleagues (1975. Br. J. Haematol. 29:537-543). Biochemical analysis of erythrocyte membrane proteins from these patients revealed, as a common abnormality, the presence of the alpha I/46-50a peptide after limited tryptic digestion of spectrin. The polymerase chain reaction was utilized to study the structure of the DNA encoding the alpha I domain of spectrin in the affected individuals. The DNA sequence of the alpha-spectrin gene encoding the region of the alpha-spectrin chain surrounding the abnormal proteolytic cleavage site was normal. We identified a point mutation causing the replacement of a highly conserved leucine residue by proline at position 207 in the alpha-spectrin chain, a site 51 residues to the amino-terminal side of the abnormal proteolytic cleavage site. Analysis of the proposed triple helical model of spectrin repeats reveals that the mutation occurs in helix 2 at a position directly opposite the abnormal proteolytic cleavage site in helix 3, making this the first report of a mutation occurring in helix 2 of a repeat in the alpha I domain of spectrin. These results add to the molecular heterogeneity of mutations associated with HE/HPP and provide further support for the proposed triple helical model of spectrin. Disruption of this proposed alpha-helical structure by helix-breaking proline substitutions may result in a functionally defective spectrin chain.

Spectrin beta-chain variant associated with hereditary elliptocytosis.

An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase. The presence of the beta'-chain was associated with a defective self-association of spectrin dimer to form tetramer as manifested by: (a) an excess of spectrin dimer in the 4 degrees C spectrin crude extract, (b) a defective self-association of the spectrin dimer in the 37 degrees C crude spectrin extracts. Gel electrophoretic analysis of the tetramer and dimer species isolated from the proband's 4 degrees C extract showed that the tetramer contained trace amounts of the beta'-chain, whereas in contrast, a large proportion of beta'-chain was present in the dimer. These results demonstrated the responsibility of the beta'-chain for the defective reassociation of spectrin dimer into tetramer. The study of this abnormal spectrin confirms the participation of spectrin beta-chain in dimer-dimer association and strongly suggests that the phosphorylation sites of the normal beta-chain are located at the end of the molecule involved in the dimer-dimer interactions.

Point mutation in the beta-spectrin gene associated with alpha I/74 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association.

alpha I/74 hereditary elliptocytosis (HE) is a subgroup of HE in which patients exhibit an impaired self-association of spectrin dimers and an abnormal proteolytic cleavage of the alpha I domain of spectrin. We studied a family in which the proband presented with a severe neonatal hemolytic anemia with poikilocytosis. Biochemical analysis of erythrocytes from the proband and his family members allowed us to ascertain a diagnosis of homozygosity for alpha I/74 HE in the proband and heterozygosity in his parents and several of their offspring. Results of polymorphism linkage analysis suggested that the defect in this family was located in beta rather than alpha spectrin. We analyzed the 3' end of the beta-spectrin gene of the proband and detected a mutation that changes a codon for alanine to one for proline. Allele-specific oligomer hybridization on slot blots of DNA from other family members confirmed the presence of the mutation only in members heterozygous for the disorder. This is the first example of a point mutation in the beta-spectrin chain that is associated with defective spectrin dimer self-association and an abnormal proteolytic cleavage of the alpha chain. Based on this finding, we propose a model for the mechanism of interaction between the alpha- and beta-spectrin chains.

Identification of the main ubiquitination site in human erythroid alpha-spectrin.

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.

Filterability and pharmacology. Effects of methylxanthine derivatives on red cell phosphorylation.

Methylxanthines interact with cyclic nucleotide-dependent phosphorylation of proteins: they are potent inhibitors of cyclic nucleotide phosphodiesterase; such that they increase cAMP level and indirectly stimulate the cAMP-dependent phosphorylation. Conversely, in the course of our studies on human red blood cell protein phosphorylation we observed that methylxanthines derivatives inhibited cAMP-independent phosphorylation by casein-kinases. Intact human red blood cells were incubated in presence or inorganic 32P with and without cAMP with various concentrations of caffeine or pentoxifylline and for various times. Haemolysis was then performed and ghosts were prepared according to Dodge et al's method. Overall phosphorylation of membrane proteins was measured and SDS-PAGE was performed with the aim to determine the phosphorylation level of separate polypeptides. We observed that cAMP-dependent phosphorylations were normal but that cAMP-independent phosphorylations were decreased proportionally to the methylxanthine derivative concentration; phosphorylation of spectrin band II and protein III were decreased. Same results were observed studying phosphorylation of ghost' proteins with gamma (32P)ATP. The mechanism of this inhibition was studied using pure casein-kinase from erythrocyte cytosol, with casein or spectrin as substrates and gamma (32P)-ATP as phosphoryl donor. The results clearly showed that caffeine and pentoxifylline were competitive inhibitors of ATP in the enzymatic phosphorylation. This would be expected since the methylxanthines are adenine analogues. If the spectrin phosphorylation is necessary to the equilibrium of cytoskeleton, methylxanthine derivatives could act on erythrocyte membrane upsetting this equilibrium.

[Clinical and biochemical study of 9 patients with hereditary elliptocytosis]. / Estudio clínico y bioquímico de 9 pacientes con eliptocitosis hereditaria.

Using clinical, functional and biochemical criteria, we studied the red blood cell membrane of nine caucasic patients from four families with hereditary elliptocytosis (HE). From a clinical point of view, seven cases were classified as compensated mild HE in whom anemia and splenomegaly are absent and reticulocytosis is slightly elevated or normal. Two cases were uncompensated mild HE with anemia, reticulocytosis, splenomegaly and erythrocytic fragmentation. (Table 1). Three individuals from one family displayed a significant reduction of protein 4.1 by polyacrylamide gel electrophoresis; one of them was uncompensated HE. The patterns of limited tryptic digestion and dimer/tetramer proportion of spectrin were normal. The study of red cell deformability by ectacytometry revealed that the cell deformability under isotonic conditions was decreased in all HE patients and the curve obtained had trapezoidal shape (Fig. 3). We found that the deformability index correlated well with the degree of anemia, but no correlation was observed between clinical findings, morphological phenotype and specific molecular etiology. According to our knowledge, this is the first report on molecular and functional studies in HE in Argentina.

Proximal giant neurofilamentous axonopathy in mice genetically engineered to resist calpain and caspase cleavage of α-II spectrin.

We use 1,2-diacetylbenzene (1,2-DAB) to probe molecular mechanisms of proximal giant neurofilamentous axonopathy (PGNA), a pathological hallmark of amyotrophic lateral sclerosis. The spinal cord proteome of rodents displaying 1,2-DAB PGNA suggests a reduction in the abundance of α-II spectrin (Spna2), a key protein in the maintenance of axonal integrity. Protein immunoblotting indicates that this reduction is due to Spna2 degradation. We investigated the importance of such degradation in 1,2-DAB PGNA. Spna2 mutant mice lacking a calpain- and/or caspase-sensitive domain (CSD), thus hypothetically resistant to 1,2-DAB, and wild-type littermates, were treated with 1,2-DAB, 35 mg/kg/day, or saline control, for 3 weeks. 1,2-DAB induced motor weakness and PGNA, irrespective of the genotype. Spna2-calpain breakdown products were not detected in mutant mice, which displayed a normal structure of the nervous system under saline treatment. Intriguingly, treatment with 1,2-DAB reduced the abundance of the caspase-specific 120-kDa Spna2 breakdown products. Our findings indicate that degradation of Spna2 by calpain- and/or caspase is not central to the pathogenesis of 1,2-DAB axonopathy. In addition, the Spna2-CSD seems to be not required for the maintenance of the cytoskeleton integrity. Our conceptual framework offers opportunities to study the role of calpain-caspase cross talk, including that of the protease degradomics, in models of axonal degeneration.

Heterogeneous phosphorylation of erythrocyte spectrin beta chain in intact cells.

Human erythrocyte spectrin is an alpha beta heterodimer which forms tetramers by self-association. This association involves the N-terminal region of the alpha chain and the C-terminal region of the beta chain. The latter contains a cluster of four phosphorylation sites (one phosphothreonine and three phosphoserine residues). The role of this phosphorylation is as yet unknown. We show in this paper that the spectrin beta chain occurs in the cell in subpopulations differing in the degree of occupancy of their phosphorylation sites: 32P peptide maps obtained by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage revealed the presence of six components with apparent molecular masses of 17.5 kDa, differing in their isoelectric points; this is most simply interpreted as reflecting the presence of six exchangeable phosphorylation sites in the spectrin beta chain, rather than four as had been supposed. When the alpha beta dimers were partly dissociated by urea, the most highly phosphorylated fraction of the beta chain was found in the undissociated dimers. This high specific activity in the undissociated dimer reflected multiple phosphorylated sites, as revealed by NTCB cleavage. The dephosphorylation or the hyperphosphorylation of spectrin beta chains did not modify the equilibrium between dissociated and undissociated spectrin dimers in the presence of urea. However, the data revealed the existence of two spectrin dimer populations in respect to phosphate turnover and spectrin dimer dissociation.

[Methylxanthines and phosphorylation of the constituents of the membrane of the human red blood cell]. / Méthylxanthines et phosphorylation des composants de la membrane du globule rouge humain.

Methylxanthines are phosphodiesterase inhibitors and are therefore capable of increasing cyclic AMP levels, thereby stimulating cyclic nucleotide-dependent protein kinases. The direct action of several xanthine derivatives on enzyme-dependent phosphorylations involving red blood cell membrane proteins was studied in vitro. Pentoxifylline and caffeine exhibited no effect on the activity of the membrane cAMP-dependent protein kinase. Conversely, methylxanthines proved capable on inhibiting cyclic nucleotide-independent protein kinases present in the membrane and cytosol. This inhibition involves competition with ATP. Comparison of the inhibitory effect of two xanthine derivatives, ie propentofylline and pentoxifylline, demonstrated significant differences. Xanthine derivatives showed no activity on red blood cell tyrosine kinase. Furthermore, three xanthines, ie caffeine, pentoxifylline and propentofylline, inhibited phosphatidylinositol kinase.

Identification of alpha-spectrin domains susceptible to ubiquitination.

Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study, we mapped the sites of ubiquitination on erythrocyte alpha-spectrin. A peptide map of digested alpha-spectrin, previously submitted to in vitro 125I-ubiquitin conjugation, revealed the presence of four distinct labeled bands with Mr 40,000, 36,000, 29,000, and 25,500. Western blotting experiments using antibodies against each alpha-spectrin domain revealed that only IgG anti-alphaIII domain recognized the 125I-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alphaV domain recognized the Mr 40,000 125I-ubiquitin-labeled peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra and tri multiubiquitin chains. Ubiquitination of the alphaIII and alphaV domains was further confirmed by anti-alpha-spectrin domain immunoaffinity chromatography. Endoprotease Lys C-digested spectrin conjugated previously to 125I-ubiquitin was incubated with antibodies against each trypsin-resistant domain of alpha-spectrin. Gamma counting of the radiolabeled antigen-antibody complexes purified by protein A chromatography showed labeling in the IgG anti-alphaIII and anti-alphaV complexes alone. Domain alphaIII is not associated with any known function, whereas domain alphaV contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiquitin in the modulation of the stability, deformability, and viscoelastic properties of the erythrocyte membrane.

[Genetic polymorphism of the alpha chain of spectrin]. / Polymorphisme génétique de la chaîne alpha de la spectrine.

On 80 black or white subjects, spectrin subunits were purified by SDS-polyacrylamide gel electrophoresis and subjected to limited alpha-chymotryptic digestion according to the Cleveland method. A genetic polymorphism exists in spectrin alpha chain and is not associated with a membranous disease. Up to now, this variant has only been detected in the black population.

[Hospital nuns attending indigent patients in the seventeenth and eighteenth centuries]. / Les soeurs hospitalières au service des pauvres malades aux XVIIe et XVIIIe siècles.

Unacknowledged, the hospital nuns deserve to be studied. Their call (or vocation), tested by the selection during novitiate, sets them into hospital environment of XVIIth and XVIIIth centuries and is rarely refuted. Whatever their own religious families, they represent the majority of nurses. In spite of some conflicts, their increasing success in the service of hospitals depends on cheap, movable, flexible, docile and polyvalent communities.

Spectrin alpha IIa variant in dominant and non-dominant spherocytosis.

Several polymorphic mutations are located on the spectrin alpha-chain; among these the variant termed alpha IIa is characterized by an acid shift in the isoelectric point of the tryptic digest peptides 46 kDa and 35 kDa. In this variant a single amino acid substitution (alanine to aspartic acid) occurred at position 972 of the spectrin alpha-chain due to a point mutation (GCT to GAT) in the DNA. This variant, which seemed very rare in normal people, could be related to the recessive form of hereditary spherocytosis (HS) and could be absent in the dominant form of the disease. We have studied the alpha IIa variant by denaturing electrophoresis of the spectrin tryptic digest peptides from 179 subjects: 46 controls, 78 patients with dominant (d) or non-dominant (nd) HS and 55 relatives of the patients. The confirmation of the results was obtained at the DNA level in 41 subjects. The frequency of the chromosome bearing the alpha IIa mutation was 7.6% in controls and higher (about 12-14%) in members of families with dHS as well ndHS. However, the family trees clearly showed that the mutation and the HS disease gene(s) were located on different chromosomes and inherited independently from each other. Furthermore, our study allows the conclusion that in most (if not all) cases of dHS, the alpha IIa the variant is not the cause, is not a marker, and does not influence the phenotypic expression of the disease.

[Hereditary elliptocytosis in West Africa: frequency and repartition of spectrin variants]. / L'elliptocytose héréditaire en Afrique de l'Ouest: fréquence et répartition des variants de la spectrine.

Hereditary Elliptocytosis (HE) is a hemolytic disorder inherited as autosomal-dominant trait and characterized by elliptically shaped erythrocytes. Preliminary studies in France have showed a high proportion of HE patients of black extraction (West Africa and Antilles). In order to confirm this prevalence, we made a systematic search for HE in West Africa: Benin, Burkina Faso, Ivory Coast, Togo. The diagnosis of elliptocytosis was established by the observation of a high percentage (greater than 70%) of characteristic regular and symmetric elliptic red cells after fixation in 0.3% glutaraldehyde saline buffer. The diagnosis of HE was confirmed by cytological studies of related members and/or the discovery of a well defined molecular variant of spectrin, the main protein of erythrocyte membrane skeleton. We found: in Abidjan centre 6 HE out of 1,000 subjects representative of main ethnic groups; in Lome Centre 6 cases out of 750 subjects originated from the South or Central areas of Togo; in Cotonou Centre 5 cases out of 1,000 subjects originated from the South area of Benin; in Bobo Dioulasso centre 6 HE out of 700 subjects. From this multicentre studies HE appears roughly 10 times more frequent in West Africa than in Europe or USA where incidence was estimated at between 2.5 and 5 cases per 10,000. Tryptic digestion of spectrin revealed that: 10 patients from different ethnic groups have the most frequent variant found in our laboratory (21 kindreds) and named spectrin alpha I/65. Five cases originated from limited areas in the South of Benin and Togo and related to closed ethnic groups have the variant Sp alpha I/46.

Identification of ubiquitinated repeats in human erythroid alpha-spectrin.

The spectrin role(s) is (are) very important for the shape and the physical properties of red cells, such as deformability and resistance to mechanical stresses. Moreover a variety of spectrin diseases are known. We have previously demonstrated [Corsi, D., Galluzzi, L., Crinelli, R. & Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935] that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo. In order to define the ubiquitinated repeats of this long protein and find out a possible function, we have produced recombinant peptides encompassing the alphaIII-, alphaIV-, alphaV- and EF hand domains of alpha-spectrin chain. These peptides were tested in in vitro ubiquitin conjugation assays and two regions susceptibles to ubiquitination were found. The first one, in the alphaIV-domain, includes the repeat 17 and the second one, in the alphaV-domain, includes the repeat 20 and a part of repeat 21. We also demonstrated that the susceptibility to ubiquitination of the alphaV-domain is reduced by interaction with the corresponding portion of beta-spectrin chain (betaIV-domain). Thus, at least ubiquitination of alphaV-domain is susceptible to cytoskeleton assembly and spectrin dimerization.