Pathologic and nonpathologic variants of the spectrin molecule in two black families with hereditary elliptocytosis

Summary Five patients with hereditary elliptocytosis (HE) from two unrelated black families were studied. The patients had prominent elliptocytosis and a decreased erythrocyte resistance to heat treatment. In one infant blood smears showed elliptocytosis and poikilocytosis; his erythrocytes fagmented at a lower temperature than those of his mother and sister, both having typical mild HE. Defective dimer-dimer association was present in all patients. Limited tryptic digestion of spectrin and subsequent analysis by one-and two-dimensional electrophoresis revealed a similar and reproducible decrease in the 80,000-dalton peptide (I domain) and the comcomitant appearance of a 46,000-dalton peptide. All the patients had the polymorphism of the spectrin II domain commonly observed in black populations. In addition, modifications relative to the III domain were detected; similar variants were found in one black control subject out of 136 and are likely related to a genetic polymorphism of the III domain. No differences were observed between the peptide patterns in the infant with poikilocytosis and those of his HE sister and mother.     

Molecular defect of truncated beta-spectrin associated with hereditary elliptocytosis. Beta-spectrin Gottingen

A large German family with autosomal dominantly inherited elliptocytosis, associated with truncated beta-spectrin missing the phosphorylated C-terminal peptide, has been described (Eber, S.W., Morris, S. A., Schroeter, W., and Gratzer, W. B. (1988) J. Clin. Invest. 81, 523-530). We have attempted to delineate the molecular defect of this abnormality at the gene level. Southern blot analyses revealed no evidence of a partial gene deletion or rearrangement. We used polymerase chain reaction (PCR) and amplified several relevant portions of the beta-spectrin gene, using genomic DNA from two different heterozygous patients. No abnormality was found in the last four exons of the beta-spectrin gene produced by PCR. Examination of the introns connecting the last four exons revealed a T to A substitution in the 5' splice site following the exon X in four of eight clones prepared from two affected individuals, but not from a normal subject of this family. To examine the effect of the T to A substitution in these patients, we made cDNA from the reticulocyte mRNA of the patient and examined its composition by PCR. Two distinct PCR fragments produced from the patient's beta-spectrin cDNA were found. One matched the predicted size for normal spectrin, while the other was 197 base pairs shorter. The mutant cDNA sequence revealed that the entire exon preceding the T to A substitution was spliced out, while the two terminal exons were preserved. The deletion of this exon resulted in a frameshift giving a different terminal amino acid (serine instead of leucine) as well as a new termination codon causing a deletion of 129 amino acids including potentially phosphorylated residues.     

Hereditary elliptocytosis in West Africa: frequency and repartition of spectrin variants

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.     

The molecular basis of the defect in phosphorylation of spectrin in human hereditary spherocytosis

The molecular basis for the depressed phosphorylation of the smaller polypeptide of spectrin (band 2) in the erythrocytes of patients suffering from hereditary spherocytosis is investigated. Comparison of healthy and spherocytic spectrin polypeptides by controlled proteolysis reveals no abnormality in the degradation pattern or in the sites of phosphorylation. It is concluded that the lesion is a consequence of a defective control of phosphorylation. The defect can be mimicked in healthy cells by the introduction of calcium into the erythrocyte and the possibility that the primary pathological lesion is a deficient control of the calcium content of the erythrocyte is discussed.     

Research on genetic abnormality in the hemolytic form of hereditary elliptocytosis with homozygosity for the spectrin alpha I/74 variant

We have undertaken to identify the spectrin gene mutation in a patient with a severe hemolytic form of Hereditary Elliptocytosis with homozygosity for the spectrin alpha I/74 variant. This variant corresponds to the presence of a 74,000 peptide which is produced during mild tryptic digestion of spectrin by cleavage at the Arginine-39 of the alpha I/80,000 domain of the spectrin alpha chain (595 amino acids). We hypothesized that the alpha I/74 mutation would be closed to the cleavage site Arg-39. A genomic library built with the patient's DNA was screened with a probe corresponding to a fragment of the alpha spectrin gene. Two clones were isolated, one being of paternal, the other of maternal origin. The subclones obtained contained the alpha spectrin gene exons 2 and 3 which encode for the first 88 amino-acids of the spectrin alpha I domain. The sequences obtained did not show any abnormality. The implications of these results are discussed.     

Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.     

Specificity of Prodan for the self-associating domain of spectrin: a molecular docking study

The hydrophobic fluorescent probe Prodan binds to the self-associating domain of spectrin with 1:1 stoichiometry. A model of the self-associating domain was generated based on its homology with other domains of spectrin. Prodan was then docked onto the model, and several sites with low interaction energy were identified. To verify whether the binding of Prodan is specific towards the self-associating domain of spectrin, it was docked on to several other domains of spectrin, having a known three-dimensional structure. Analysis of the docking results suggests that the binding of Prodan to the self-associating domain of spectrin will involve hydrophobic and hydrophilic groups of Prodan. The results clearly indicate the preference of Prodan for a particular binding site of the self-associating domain.     

Structure of human erythrocyte spectrin. II. The sequence of the alpha-I domain

The complete sequence of 595 amino acids of the alpha-I domain of human erythrocyte spectrin has been determined. Peptides derived from three different protease cleavages were purified using high performance liquid chromatography and subjected to automated amino acid sequence analysis. These data along with sequences of the cyanogen bromide and large tryptic peptides (Speicher, D.W., Davis, G., Yurchenco, P.D., and Marchesi, V.T. (1983) J. Biol. Chem. 258, 14931-14937) represent most or all of the sequence of spectrin alpha-I. The single remaining ambiguity is the precise termination of the COOH terminus of the alpha-I domain. The sequence data suggest that the 595 residues presented here represent the complete sequence of the alpha-I domain, but the apparent size of the COOH-terminal CNBr fragment suggests the existence of an additional 38 residues at the end of the domain. The sequence of the alpha-I domain contains a single type of internal homology composed of multiple 106-amino acid repeats consistent with the occurrence of multiple gene duplications during the course of spectrin evolution. The only portion of the alpha-I sequence which does not appear to contain this sequence repeat is the segment containing the NH2-terminal 17 residues. This unique segment may be part of the oligomer binding site. No disulfide bonds appear to be involved in the structure of alpha-I and cysteine is not highly conserved. Calculations of secondary structure suggest the presence of short helices which fold into triple helical segments approximately 50 A in length. There is little beta sheet structure. A model of spectrin structure incorporating the repeat unit and proposed secondary structure is presented. A computer search of alpha-I sequence with the National Biomedical Research Foundation database of 2145 protein sequences did not detect any significant relationships. Spectrin is apparently the first member of a new class of proteins to be structurally characterized.     

Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA.

Protein interactions with double-stranded RNA (dsRNA) are critical for many cell processes; however, in contrast to protein-dsDNA interactions, surprisingly little is known about the molecular basis of protein-dsRNA interactions. A large and diverse class of proteins that bind dsRNA do so by utilizing an approximately 70 amino acid motif referred to as the dsRNA-binding domain (dsRBD). We have determined a 1.9 A resolution crystal structure of the second dsRBD of Xenopus laevis RNA-binding protein A complexed with dsRNA. The structure shows that the protein spans 16 bp of dsRNA, interacting with two successive minor grooves and across the intervening major groove on one face of a primarily A-form RNA helix. The nature of these interactions explains dsRBD specificity for dsRNA (over ssRNA or dsDNA) and the apparent lack of sequence specificity. Interestingly, the dsRBD fold resembles a portion of the conserved core structure of a family of polynucleotidyl transferases that includes RuvC, MuA transposase, retroviral integrase and RNase H. Structural comparisons of the dsRBD-dsRNA complex and models proposed for polynucleotidyl transferase-nucleic acid complexes suggest that similarities in nucleic acid binding also exist between these families of proteins.     

On the biochemical basis of hereditary fructose intolerance.

In hereditary fructose intolerance it was found that in addition to an increased K_m value for Fru-1-P, the K_m of aldolase for Fru-1,6-P₂ was also increased. Furthermore, human phosphorylase $\text{a}$ was found to be inhibited by Fru-1-P in a non-competitive way.     

Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands.

Calcium is a universally employed cytosolic messenger in eukaryotic cells. Most of the proteins that bind signalling calcium are members of the calmodulin superfamily and share two or more helix-loop-helix motifs known as EF-hands. A model, based on structure comparison of different domains and supported by preliminary NMR data, has suggested that EF-hands involved in signal transduction undergo a major conformational change upon calcium binding from a 'closed' to an 'open' state allowing protein-protein interaction. We have determined the solution structures of the EF-hand pair from alpha-spectrin in the absence and in the presence of calcium. The structures are in the closed and open conformation respectively, providing a definite experimental proof for the closed-to-open model. Our results allow formulation of the rules which govern the movement induced by calcium. These rules may be generalized to other EF-hands since the key residues involved are conserved within the calmodulin family.     

Abnormal binding of spectrin to the membrane of erythrocytes in some cases of hereditary spherocytosis

In two cases of hereditary spherocytosis that we have examined, spectrin was bound abnormally tightly to the erythrocyte membrane, and could not be released by low ionic strength dialysis. This type of behaviour occurs in normal red cells only after heating above 50 degrees C. It appears that some cases of spherocytosis may be due to the presence of a protein which is abnormally temperature sensitive.     

Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.     

Molecular basis of Wnt activation via the DIX domain protein Ccd1

The Wnt signaling plays pivotal roles in embryogenesis and cancer, and the three DIX domain-containing proteins, Dvl, Axin, and Ccd1, play distinct roles in the initiation and regulation of canonical Wnt signaling. Overexpressed Dvl has a tendency to form large polymers in a cytoplasmic punctate pattern, whereas the biologically active Dvl in fact forms low molecular weight oligomers. The molecular basis for how the polymeric sizes of Dvl proteins are controlled upon Wnt signaling remains unclear. Here we show that Ccd1 up-regulates canonical Wnt signaling via acting synergistically with Dvl. We determined the crystal structures of wild type Ccd1-DIX and mutant Dvl1-DIX(Y17D), which pack into "head-to-tail" helical filaments. Structural analyses reveal two sites crucial for intra-filament homo- and hetero-interaction and a third site for inter-filament homo-assembly. Systematic mutagenesis studies identified critical residues from all three sites required for Dvl homo-oligomerization, puncta formation, and stimulation of Wnt signaling. Remarkably, Ccd1 forms a hetero-complex with Dvl through the "head" of Dvl-DIX and the "tail" of Ccd1-DIX, depolymerizes Dvl homo-assembly, and thereby controls the size of Dvl polymer. These data together suggest a molecular mechanism for Ccd1-mediated Wnt activation in that Ccd1 converts latent polymeric Dvl to a biologically active oligomer(s).     

Molecular basis for interaction between Icap1 alpha PTB domain and beta 1 integrin.

Icap1 alpha is a 200-amino acid protein that binds to the COOH-terminal 13 amino acids ((786)AVTTVVNPKYEGK(798)) of the integrin beta(1) subunit. Alanine scanning mutagenesis of this region revealed that Val(787), Val(790), and (792)NPKY(795) are critical for Icap1 alpha binding. The NPXY motif is a known binding substrate for phosphotyrosine binding (PTB) domain proteins. The sequences of Icap1 alpha, residues 58--200, and the beta(1) integrin, residues 786-797, were aligned to the available PTB-peptide structures to generate a high quality structural model. Site-directed mutagenesis showed that Leu(135), Ile(138), and Ile(139) of Icap1 alpha, residues predicted by the model to be in close proximity to (792)NPKY(795), and Leu(82) and Tyr(144), residues expected to form a hydrophobic pocket near Val(787), are required for the Icap1 alpha-beta(1) integrin interaction. These findings indicate that Icap1 alpha is a PTB domain protein, which recognizes the NPXY motif of beta(1) integrin. Furthermore, our date suggest that an interaction between Val(787) and the hydrophobic pocket created by Leu(82) and Tyr(144) of Icap1 alpha forms the basis for the specificity of Icap1 alpha for the beta(1) integrin subunit.     

Sequence similarity of the amino-terminal domain of Drosophila beta spectrin to alpha actinin and dystrophin

We used chicken alpha spectrin as a ligand probe to isolate Drosophila beta spectrin cDNA sequences from a lambda gt11 expression library. Analysis of 800 residues of deduced amino acid sequence at the amino- terminal end revealed a strikingly conserved domain of integral of 230 residues that shows a high degree of sequence similarity to the amino- terminal domains of alpha actinin and dystrophin. This conserved domain constitutes a new diagnostic criterion for spectrin-related proteins and allows the known properties of one of these proteins to predict functional properties of the others. The conservation of the amino- terminal domain, and other regions in spectrin, alpha actinin, and dystrophin, demonstrates that a common set of domains were linked in different combinations through evolution to generate the distinctive members of the spectrin superfamily.     

Molecular basis for differential nucleotide binding of the nucleotide-binding domain of ABC-transporter CvaB

The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC-transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP and, indeed, highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide-binding domain predicted that the aromatic stacking region of CvaB (Y501DSQ loop) had a role in the differential binding of nucleotides, and Ser503 and Gln504 provided potential hydrogen bonds to GTP but not to ATP. Site-directed mutagenesis of the Y501DSQ loop, mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature effect assays on nucleotide binding and hydrolysis further indicated that the double mutant protein had largely eliminated the difference for substrates ATP and GTP, and behaved more similarly to the NBD of typical ABC-transporter HlyB. Therefore, we conclude that Ser503 and Gln504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference.