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V T Marchesi

Publications and source records attributed to V T Marchesi.

At least 19 recordsLinked to original sources
PubMed1990

Full-length sequence of the cDNA for human erythroid beta-spectrin.

Spectrin is the major molecular consituent of the red cell membrane skeleton. We have isolated overlapping human erythroid beta-spectrin cDNA clones and determined 6773 base pairs of contiguous nucleotide sequence. This includes the entire coding sequence of beta-spectrin. The sequence translates into a 2137 amino acid, 246-kDa peptide. beta-Spectrin is found to consist of three distinct domains. Domain I, at the N terminus, is a 272-amino acid region lacking resemblance to the spectrin repetitive motif. Sequences in this region exhibit striking sequence homology, at both nucleotide and amino acid levels, to the N-terminal "actin-binding" domains of alpha-actinin and dystrophin. Between residues 51 and 270 there is 55% amino acid identity to human dystrophin, with only four single amino acid gaps in alignment. Domain II consists of 17 spectrin repeats. Several sequence variations are observed in typical repeat structure. Homology to alpha-actinin extends beyond domain I into the N-terminal portion of domain II. Domain III, 52 amino acid residues at the C terminus, does not adhere to the spectrin repeat motif. Combining knowledge of spectrin primary structure with previously reported functional studies, it is possible to make several inferences regarding structure/function relationships within the beta-spectrin molecule.

Amino Acid Sequence↗
PubMed1990

The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.

Actinin↗
PubMed1990

Heterogeneity of mRNA and protein products arising from the protein 4.1 gene in erythroid and nonerythroid tissues.

Immunologically cross-reactive isoforms of the cytoskeletal element protein 4.1 have been identified in many tissues in which they exhibit heterogeneity of molecular weight, abundance, and intracellular localization. To examine the basis for isoform production in erythroid and nonerythroid tissues, we have compared the structure and expression of cDNAs isolated from human erythroid and nonerythroid sources. We have encountered cDNAs representing many distinct mRNA sequences. These exhibit complete nucleotide sequence homology along most of their lengths. Differences were confined to five sequence blocks designated Motifs I-V, which were present or absent in each mRNA moiety. Motif I was expressed only in erythroid cells; it encodes 21 amino acids in a well-characterized spectrin/actin binding domain. Motif II, located near the COOH terminus of the 80-kD "erythroid" protein 4.1 molecule is present in the vast majority of transcripts from both erythroid and nonerythroid cells. Motifs IV and V alter the 5' untranslated region: simultaneous insertion of Motif IV and deletion of Motif V in the untranslated region inserts a new initiator methionine and establishes a contiguous open reading frame encoding a novel 135-kD protein 4.1 molecule. By immunochemical analysis we have identified the longer isoform in cells. Our results are most consistent with tissue-specific alternative mRNA splicing of transcripts of the protein 4.1 gene to yield numerous isoforms. These isoforms exhibit tissue specificity and alter strategic portions of the molecule. Moreover, we describe a novel high molecular weight form of protein 4.1 that arises by splicing events which allow translation at an upstream site.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations.

Protein 4.1 (80 kD) interacts with spectrin and short actin filaments to form the erythrocyte membrane skeleton. Mutations of spectrin and protein 4.1 are associated with elliptocytosis or spherocytosis and anemia of varying severity. We analyzed two mutant protein 4.1 molecules associated with elliptocytosis: a high molecular weight 4.1 (95 kD) associated with mild elliptocytosis without anemia, and a low molecular weight 4.1 (two species at 68 and 65 kD) associated with moderate elliptocytosis and anemia. 4.1(95) was found to contain a approximately 15-kD insertion adjacent to the spectrin/actin binding domain comprised, at least in part, of repeated sequence. 4.1(68/65) was found to lack the entire spectrin-actin binding domain. The mechanical stability of erythrocyte membranes containing 4.1(95) was identical to that of normal membranes, consistent with the presence of an intact spectrin-actin binding domain in protein 4.1. In contrast, membranes containing 4.1(68/65) have markedly reduced mechanical stability as a result of deleting the spectrin-actin binding domain. The mechanical stability of these membranes was improved following reconstitution with normal 4.1. These studies have thus enabled us to establish the importance of the spectrin-actin binding domain in regulating the mechanical stability of the erythrocyte membrane.

Actins↗
PubMed1989

Characterization of the calmodulin-binding site of nonerythroid alpha-spectrin. Recombinant protein and model peptide studies.

An important function of the mammalian nonerythroid alpha-spectrin chain (alpha-fodrin) that distinguishes it from the closely related erythroid isoform is its ability to bind calmodulin. By analysis of a series of deleted recombinant spectrin fusion proteins, we have identified a region in the nonerythroid alpha chain involved in calcium-dependent binding of calmodulin. The region is distinctive in that the sequence is absent from the homologous domain of the erythroid alpha chain and diverges from the normal internal repeat structure observed throughout other spectrins. In order to determine limits of this functional site, a synthetic peptide as small as 24 residues was shown to compete with either recombinant or brain alpha-spectrin in binding to calmodulin. The active peptide, which was derived from a segment between repeats 11 and 12, was composed of the following sequence: Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala-Thr-Phe-Asn - Ser-Ile-Lys-Glu. Comparison of this sequence with functional sites in other diverse calcium-dependent calmodulin-binding proteins has revealed a structural motif common to all of these proteins, namely clusters of hydrophobic residues interspersed with basic residues. When folded into alpha-helical conformations, these binding sites are predicted to form amphipathic structures.

Amino Acid Sequence↗
PubMed1989

Synthetic peptides mimic the assembly of transmembrane glycoproteins.

The composition of the intramembranous domains of many receptors are remarkably uniform, yet there is evidence that many transmembrane proteins associate together to form specific noncovalent homo- or heterocomplexes within the membrane. We have synthesized peptides corresponding to transmembrane domains of glycophorin A, glycophorin C, and the interleukin 2-receptor Tac antigen to study the interactions between transmembrane domains in vitro. Synthetic transmembrane glycophorin A peptide formed a complex with native glycophorin and glycoproteins of erythrocyte and K562 cell membranes that was reversible, specific, and could be demonstrated in a natural bilayer system in the absence of detergents. Synthetic glycophorin C and interleukin 2-receptor Tac antigen transmembrane peptides, although similar in amino acid composition, did not interact with glycophorin and did not inhibit the binding of the synthetic glycophorin A transmembrane peptide to native glycophorin. It is proposed that the transmembrane segments of receptor proteins contain not only the structural information necessary for insertion and anchoring but specific binding sites that mediate interactions between transmembrane glycoproteins.

Amino Acid Sequence↗
PubMed1989

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.

Amino Acid Sequence↗
PubMed1988

Erythrocyte membrane skeleton phosphoproteins: identification of two unrelated phosphoproteins in band 4.9.

Human erythrocyte membrane band 4.9 is phosphorylated by several erythrocyte protein kinases. Chromatography of erythrocyte membrane skeleton proteins on DEAE-Sephacel produces two proteins with relative mobilities, on gel electrophoresis, similar to that of band 4.9. The first, with a molecular mass of 49 kDa, is quite basic (pI greater than 8) while the second, 50.5 kDa, is slightly acidic (pI = 6.2). Comparative two-dimensional peptide mapping reveals that both proteins are present in band 4.9 on one-dimensional gels of total erythrocyte membrane proteins and membrane skeleton proteins. The 49 kDa protein, but not the 50.5 kDa protein, binds to actin filaments in a sedimentation assay. In intact erythrocytes metabolically labeled with [32P]orthophosphate, the 49 kDa protein is phosphorylated by protein kinase C, cAMP-dependent protein kinase, and protein kinases which are active in the absence of exogenous kinase activators. In contrast, the 50.5 kDa protein is phosphorylated by protein kinase C but not by the other protein kinases examined. Finally, two-dimensional peptide mapping was employed to compare the 49 kDa protein and a 57 kDa protein which copurifies with, and has many characteristics of, the 49 kDa protein. Significant similarities were found in both 125I-labeled chymotryptic peptide maps and 32P-labeled tryptic peptide maps, suggesting that the 49 kDa and 57 kDa proteins are closely related.

Blood Proteins↗
PubMed1988

Tissue-specific analogues of erythrocyte protein 4.1 retain functional domains.

Analogues of the human erythroid membrane skeletal component protein 4.1 have been identified in perfused rat tissues and human T and B lymphocyte cell lines. olyclonal antibodies were used which are specific for all domains of protein 4.1, the spectrin-actin-promoting 8-Kd peptide, the membrane-binding 30-Kd domain, and the 50-Kd domain. Antibody reactivity, by Western blotting of tissue homogenates, shows reactivity with proteins varying in molecular weight from 175 Kd to 30 Kd. Further, these protein 4.1 analogues appear to be expressed in a tissue-specific fashion. Of the analogues detected there appear to be at least three classes: analogues containing all erythroid protein 4.1 domains, analogues containing all domains but with modified antigenic epitopes, and analogues containing only some domains. Chemical cleavage at cysteine linkages indicates that in analogues containing the 30-Kd region the location of cysteine is highly conserved. This datum suggests that in nonerythroid 4.1 isoforms of higher molecular weight the additional protein mass is added to the amino terminal end (30 Kd end).

Animals↗
PubMed1988

Expression of specific isoforms of protein 4.1 in erythroid and non-erythroid tissues.

Protein 4.1 in red cells is an important submembrane linking protein that binds to spectrin actin complexes at one end of its structure and to transmembrane proteins, such as glycophorin, at the other. Protein 4.1 thus contributes to the strength and flexibility of the erythrocyte membrane, a fact dramatically exemplified by the appearance of hereditary hemolytic anemias in patients with absent or abnormal protein 4.1. Recently, protein 4.1 forms have been discovered in many non-erythroid tissues. Their intracellular locations raise the possibility that these isoforms might have different functions. We have thus conducted comparative analysis of erythroid and non-erythroid protein 4.1 forms by cloning and sequencing erythroid and lymphoid protein 4.1 cDNAs. The lymphoid protein 4.1 isoforms exhibit at least five nucleotide sequence motifs that appear to be either inserted or deleted relative to the erythroid mRNA sequence by alternative splicing of a common mRNA precursor. One of these motifs, located within the spectrin-actin binding domain, is found only in erythroid cells and is specifically produced during erythroid cell maturation. The selective expression of this alternatively spliced mRNA during erythroid maturation implies the existence of a lineage specific splicing mechanism whose activity is triggered by terminal maturation. Two motifs alter the 5' untranslated region of the "prototypical" erythroid mRNA in such a way as to permit synthesis of a novel larger isoform. This form appears to localize preferentially in the nucleus. We thus conclude that a single gene gives rise to multiple protein 4.1 isoforms with potentially diverse locations and functions.

Base Sequence↗
PubMed1988

Selective expression of an erythroid-specific isoform of protein 4.1.

We have conducted comparative analysis of nucleotide sequences encoding erythroid and lymphoid protein 4.1 isoforms. The lymphoid protein 4.1 isoforms exhibit several nucleotide sequence motifs that appear to be either inserted into or deleted from the mRNA sequence by alternative splicing of a common mRNA precursor. One of these motifs, located within the spectrin-actin binding domain, is found only in erythroid cells and is specifically produced during erythroid cell maturation. The selective expression of the alternatively spliced mRNA during erythroid maturation implies the existence of a lineage-specific splicing mechanism whose activity is triggered by terminal maturation.

Amino Acid Sequence↗
PubMed1988

Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.

The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of the cDNA for human erythrocyte beta-spectrin.

Overlapping cDNA clones, totaling 3.3 kilobases (kb) in length, which encode over 50% of the human erythrocyte beta-spectrin subunit, were isolated by antibody screening of a lambda gt11 expression library constructed from human fetal liver mRNA. The amino acid sequence of the C-terminus of beta-spectrin was derived. The size of beta-spectrin mRNA in human erythroleukemia cells was found to be 7.5 kb. Erythrocyte beta-spectrin is encoded by a gene located on human chromosome 14, as determined by cDNA hybridization to human X mouse somatic cell hybrids.

Amino Acid Sequence↗
PubMed1987

Mutant forms of spectrin alpha-subunits in hereditary elliptocytosis.

Two variant spectrins have been described in hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP). Both are characterized by increased susceptibility of the alpha I (N-terminal) 80-kD domain to mild tryptic digestion, yielding peptides of 46-50 or 65-68 kD (T50a and T68 in our terminology). In this report we add a third unstable spectrin alpha I domain found in three kindreds with HE; alpha IT80 in this type of spectrin is cleaved by mild tryptic digestion to a 50-kD peptide (T50b) distinguished from T50a by its more basic isoelectric point. All three spectrins show impaired self-association to form oligomers. Intermediate tryptic peptides of the three unstable alpha I domains from HE spectrins were characterized by monoclonal immunoblotting and I125 limit peptide mapping and affinity purified using polyclonal anti-alpha IT80. Partial amino acid sequences of alpha I domain peptides were obtained from two unrelated patients for each of the three variant spectrins. T50a results from cleavage at arginine 250 or lysine 252 of alpha IT80; a proline replaced the normal leucine or serine at residues 254 and 255, respectively. T50b and a 19-kD peptide result from cleavage at arginine 462 or arginine 464; a proline replaced the normal residue 465 (in T19b) in one of the two patients studied. T68 results from cleavage at arginine 131. In both 68-kD peptides examined, a leucine is inserted at residue 150. The relationship of the sequence changes to the new tryptic cleavages, to the current model of alpha I domain structure, and to defective spectrin self-association is discussed.

Amino Acid Sequence↗
PubMed1986

Structure of the spectrin-actin binding site of erythrocyte protein 4.1.

The complete primary structure of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations has been determined. The sequence of this domain, which contains 67 amino acids and has a molecular mass of 8045 daltons, has been obtained by NH2-terminal sequence analysis of an 8-kDa chymotryptic peptide, three endoproteinase lysine C-cleaved peptides and two peptides obtained by Staphylococcus aureus protease V8 cleavage. All peptides including the 8-kDa domain peptide were purified by reverse-phase high performance liquid chromatography. Antibodies against two different synthetic peptides of the 8-kDa domain are able to inhibit the association between protein 4.1, spectrin, and F-actin, corroborating that the 8-kDa domain is responsible for the formation of a ternary complex. A computer search of the 8-kDa sequence with the National Biomedical Research Foundation database did not detect any significant homologies to known sequences. Protein 4.1 is not related to any known proteins and may represent a new protein superfamily.

Actins↗
PubMed1986

A calmodulin and alpha-subunit binding domain in human erythrocyte spectrin.

Human erythrocyte spectrin binds calmodulin weakly under native conditions. This binding is enhanced in the presence of urea. The site responsible for this enhanced binding in urea has now been shown to reside in a specific region of the spectrin beta-subunit. Cleavage of spectrin with trypsin, cyanogen bromide or 2-nitro-5-thiocyanobenzoic acid generates fragments of the molecule which retain the ability to bind calmodulin under denaturing conditions. The origin of these fragments, identified by two-dimensional peptide mapping, is the terminal region of the spectrin beta-IV domain. The smallest peptide active in calmodulin binding is a 10 000 Mr fragment generated by cyanogen bromide cleavage. Only the intact 74 000 Mr fragment generated by trypsin (the complete beta-IV domain) retains the capacity to reassociate with the isolated alpha-subunit of spectrin. The position of a putative calmodulin binding site near a site for subunit-subunit association and protein 4.1 and actin binding suggests a possible role in vivo for calmodulin regulation of the spectrin-actin membrane skeleton or for regulation of subunit-subunit associations. This beta-subunit binding site in erythrocyte spectrin is found in a region near the NH2-terminus at a position analogous to the alpha-subunit calmodulin binding site previously identified in a non-erythroid spectrin by ultrastructural studies.

Binding Sites↗
PubMed1986

Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations.

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability of intact 4.1 to promote the binding of spectrin to F-actin. This complex-promoting activity was found to reside in an 8-kDa peptide which was fully functional when dissociated from other protein 4.1-derived peptides, indicating that noncovalent complexes of multiple peptides were not essential for activity. The 8-kDa peptide was incorporated into a ternary complex with spectrin and F-actin in approximately stoichiometric amounts. Amino acid composition and two-dimensional peptide mapping show that the 8-kDa active peptide is located within the 10-kDa region of protein 4.1 which contains a cAMP-dependent phosphorylated site.

Actins↗