Tropomyosin Sequences Research Articles

Toward Consensus Epitopes B and T of Tropomyosin Involved in Cross-Reactivity across Diverse Allergens: An In Silico Study.

Tropomyosin (TM) is a pan-allergen with cross-reactivity to arthropods, insects, and nematodes in tropical regions. While IgE epitopes of TM contribute to sensitization, T-cell (MHC-II) epitopes polarize the Th2 immune response. This study aimed to identify linear B and T consensus epitopes among house dust mites, cockroaches, Ascaris lumbricoides, shrimp, and mosquitoes, exploring the molecular basis of cross-reactivity in allergic diseases. Amino acid sequences of Der p 10, Der f 10, Blo t 10, Lit v 1, Pen a 1, Pen m 1, rAsc l 3, Per a 7, Bla g 7, and Aed a 10 were collected from Allergen Nomenclature and UniProt. B epitopes were predicted using AlgPred 2.0 and BepiPred 3.0. T epitopes were predicted with NetMHCIIpan 4.1 against 10 HLA-II alleles. Consensus epitopes were obtained through analysis and Epitope Cluster Analysis in the Immune Epitope Database. We found 7 B-cell epitopes and 28 linear T-cell epitopes binding to MHC II. A unique peptide (residues 160-174) exhibited overlap between linear B-cell and T-cell epitopes, highly conserved across tropomyosin sequences. These findings shed light on IgE cross-reactivity among the tested species. The described immuno-informatics pipeline and epitopes can inform in vitro research and guide synthetic multi-epitope proteins' design for potential allergology immunotherapies. Further in silico studies are warranted to confirm epitope accuracy and guide future experimental protocols.

Molecular Characterization of Tropomyosin and Its Potential Involvement in Muscle Contraction in Pacific Abalone.

Tropomyosin (TPM) is a contractile protein responsible for muscle contraction through its actin-binding activity. The complete sequence of TPM in Haliotis discus hannai (Hdh-TPM) was 2160 bp, encoding 284 amino acids, and contained a TPM signature motif and a TPM domain. Gene ontology (GO) analysis based on the amino acid sequence predicted Hdh-TPM to have an actin-binding function in the cytoskeleton. The 3D analysis predicted the Hdh-TPM to have a coiled-coil α-helical structure. Phylogenetically, Hdh-TPM formed a cluster with other TPM/TPM1 proteins during analysis. The tissue-specific mRNA expression analysis found the higher expression of Hdh-TPM in the heart and muscles; however, during embryonic and larval development (ELD), the higher expression was found in the trochophore larvae and veliger larvae. Hdh-TPM expression was upregulated in fast-growing abalone. Increasing thermal stress over a long period decreased Hdh-TPM expression. Long-term starvation (>1 week) reduced the mRNA expression of Hdh-TPM in muscle; however, the mRNA expression of Hdh-TPM was significantly higher in the mantle, which may indicate overexpression. This study is the first comprehensive study to characterize the Hdh-TPM gene in Pacific abalone and to report the expression of Hdh-TPM in different organs, and during ELD, different growth patterns, thermal stress, seasonal changes, and starvation.

Identification of linear epitopes and their major role in the immunoglobulin E-binding capacity of tropomyosin from Alectryonella plicatula.

Tropomyosin (TM) is an important allergen in molluscans. However, there was a lack of information about TM as an allergen in oysters. TM was purified and identified from Alectryonella plicatula (ATM), and its primary sequence was cloned and encoded with 284 amino acids (AAs). Chemical denaturants were used to destroy the structure to confirm that linear epitopes played a major role in the immunoglobulin E-binding capacity of ATM. Subsequently, nine linear epitopes were identified using a serological test. The peptide with AA27-41 was regarded as the key epitope because it could be recognized strongly by most sera of oyster-sensitive individuals in comparison to other epitope peptides. Finally, the epitopes and the primary sequence of TM among shellfish were aligned to find the two conserved epitopes (AA117-132 and AA164-178) in oyster, octopus, abalone, scallop, clam, shrimp, and crab. Overall, these data provide a foundation for the allergenicity and cross-reactivity of TM.

Immunochemical analysis of pepsin-digested fish tropomyosin

A critical step in determining the allergenicity of a food protein is to study the gastrointestinal digestion resistance, which is an essential characteristic of food allergens due to the preservation of their protein structure and immunoglobulin (Ig) E epitopes. In this study, the immunochemical properties of tropomyosin peptide fragments from Atlantic salmon (Salmo salar) and flathead grey mullet (Mugil cephalus) resulted from pepsin digestion were analyzed. To identify the allergens from salmon and mullet, their IgE- and IgG-binding proteins/peptides were studied using fish-allergic human sera and anti-fish protein monoclonal and polyclonal antibodies. From our results, as the major fish allergen, parvalbumin from both species was recognized in all tested human sera. Tropomyosin-bound IgE was identified in 71% of the tested sera. Antigenicity of intact tropomyosin decreased as a function of pepsin digestion time. The amino acid sequence of mullet tropomyosin was analyzed using a tandem mass spectrometer. Two IgE epitopes on Atlantic salmon tropomyosin alpha-1 were mapped. Overall, tropomyosin was identified as one of the major IgE-binding proteins in both fish species; however, further studies are required to determine the clinical significance of fish tropomyosin as an allergen due to its high homology with human tropomyosin.

Functional effects of substitutions I92T and V95A in actin-binding period 3 of tropomyosin

Tropomyosin polymerizes along actin filaments and together with troponin regulates muscle contraction in a Ca-dependent manner. Actin-binding periods are homologous residues, which repeat along tropomyosin sequence, form tropomyosin-actin interface and determine regulatory functions. To learn how period 3 is involved in tropomyosin functions we examined effects of two mutations in Tpm1.1, I92T and V95A, which have been linked to dilated and hypertrophic cardiomyopathies characterized respectively by hyper- and hypocontractile phenotypes. In this work the functional consequences of both mutations were studied in vitro by using actin thin filaments reconstituted in the presence of mutant Tpm1.1 homodimers carrying the substitutions in both tropomyosin chains, Tpm1.1 heterodimers with substitution only in one Tpm1.1 chain, and Tpm1.1/Tpm2.2 heterodimers with substitution in Tpm1.1 chain and wild type Tpm2.2 in the second chain. The presence of the substitution I92T decreased the tropomyosin affinity for actin, abolished Ca2+-dependent activation of the actomyosin ATPase, decreased the sensitivity of the tropomyosin-troponin complex to subsaturating Ca2+ concentrations and reduced the cooperativity of the myosin-induced transition of the thin filament to a fully active state. The substitution V95A had opposite effects: increased actin affinity, increased the actomyosin ATPase activity above the level observed for wild type Tpm and increased cooperativity of myosin-induced activation of the thin filaments reconstructed with homo- and heterodimers of tropomyosin. Substitutions I92T and V95A were dominant, but the formation of heterodimers modified the effects observed for homodimers.

Genomic Organization of Repetitive DNAs and Differentiation of an XX/XY Sex Chromosome System in the Amazonian Puffer Fish, Colomesus asellus (Tetraodontiformes)

The genus Colomesus is the sole representative of the family Tetraodontidae in the Amazon region. Here, Colomesus asellus was analyzed using conventional and molecular cytogenetic protocols. Its diploid chromosome number is 2n = 46 with 12 meta-, 10 submeta-, 16 subtelo-, and 8 acrocentric chromosomes and a fundamental number of FN = 84. An XX/XY sex chromosome system was identified. Mapping of 18S rDNA correlated with the nucleolus organizer regions (Ag-NORs) in the short arms of the 2 X chromosomes in females and in the Y chromosome in males. C-banding revealed heterochromatin in the centromeric regions of all chromosomes, except for pair 3. Prominent sex chromosome-specific heterochromatin amplification was observed, covering the short arms of the Y chromosome almost entirely. FISH with telomeric and tropomyosin (tpm1) sequences, respectively, revealed terminal signals in all chromosomes. The analysis of extended DNA fibers confirmed the colocalization and the interspersed pattern of the telomeric and tpm1 sequences. Thus, this study highlights the remarkable evolutionary dynamism presented by the Amazonian puffer fish regarding the differentiation of a heteromorphic XY sex chromosome system and a particular sex-specific amplification of rDNA sites. This is the first record of such an association in the Tetraodontidae family.

IgE Cross-Reactivity Between Shellfish and Diverse Potential Food Insects

Is observed IgE cross-reactivity of insect and shellfish tropomyosins explained by sequence and/or structural characteristics and if so can we use this to predict likely reactivity? We analyzed cross-reactivity patterns of shellfish and seven insect species using sera from 14 shellfish allergic individuals using IgE immunoblotting. We obtained protein sequences either from public databases (where available) or from translation of novel insect tropomyosin genes cloned in our lab. Relevence of both existing and novel tropomyosin sequences was demonstrated by quantitative protein mass spectrometry. Distinct patterns of cross-reactivity were observed, with three edible insect species consistently displaying lower binding of IgE. This pattern was not due to overall sequence homology. These results will be discussed with reference to differences in localized protein sequence motifs and other phyisco-chemical protein properties. Edible insects vary in the capacity of major allergens to bind shrimp allergic IgE, and therefore potentially elicit allergic reactions. This information is essential for the successful risk assessment of edible insects as potential food allergens.

The Occurrence of Sequences Identical with Epitopes from the Allergen Pen a 1.0102 Among Food and Non-Food Proteins

Tropomyosins and their fragments belong to the seafood allergens [Jedrychowski et al., 2008; Hajeb & Selamat, 2012; Kumar et al., 2012]. Their allergenic properties are usually attributed to invertebrate proteins and peptides derived from them [Kumar et al., 2012]. Few years ago vertebrate tropomyosins were regarded as non-allergenic proteins due to their susceptibility to hydrolysis by pepsin (EC 3.4.23.1; ID A01.001 in the MEROPS database of proteolytic enzymes) or absence of epitopes characteristic for invertebrate tropomyosins. On the other hand, the number of protein sequences recorded in databases is growing rapidly and it is possible that the above-mentioned criteria will not be fulfi lled by new vertebrate tropomyosin sequences. The fi rst allergenic tropomyosin of vertebrate origin – protein from fi sh Oreochromis mossambicus (Ore m 4.0101, UniProt accession No: K4PEK4, Allergome code 10146) has been recently described [Liu et al., 2013] on the basis of experimental results. Some vertebrate tropomyosins (e.g. human) are annotated in Allergome database as in silico predicted allergens on the basis of their amino acid sequences. Tropomyosin sequences are highly conserved and some of them were known to contain common sequential epitopes.

Determination of microheterogeneous substitution in shrimp tropomyosin and its effect on IgE-binding capacity

Tropomyosin (TM), the major shrimp allergen that belongs to a highly conserved protein family, has frequently been investigated owing to its high rate of human consumption. In this study, the sequence microheterogeneity of TMs, irrespective of the organism and differences in the N- and C-termini, from different shrimp species was analyzed. The TM sequences were analyzed using bioinformatics tools and confirmed by dot-blot using human serum. The results showed that all 13 shrimp species share high mutual TM sequence identity values, ranging from 85.5 to 99.7 %, and 60 cases of sequence microheterogeneity were identified in shrimp TM, with a sequence identity ranging from 90.1 to 99.7 %. A total of 719 unique amino acid residue substitutions (that affect 43 residues, 15 % of 284 residues) involving sequence microheterogeneity were characterized, in which 214 substitutions involving 15 residues were located in the epitope regions. About 45 % of these substitutions occurred in the TM sequences that are reported allergens, whereas 43 % of the substitutions occurred overall for the allergenic and nonallergenic TMs. Dot-blot immunoassay revealed that single microheterogeneous mutation of allergic peptides resulted in significant changes in the IgE-binding capacity of the peptides. Overall, microheterogeneous mutations of TM occurred in different shrimp species. These mutations can potentially induce changes in the IgE-binding capacity and might be responsible for variations in the hypersensitivity to different shrimp species.

Structure and flexibility of the tropomyosin overlap junction

To be effective as a gatekeeper regulating the access of binding proteins to the actin filament, adjacent tropomyosin molecules associate head-to-tail to form a continuous super-helical cable running along the filament surface. Chimeric head-to-tail structures have been solved by NMR and X-ray crystallography for N- and C-terminal segments of smooth and striated muscle tropomyosin spliced onto non-native coiled-coil forming peptides. The resulting 4-helix complexes have a tight coiled-coil N-terminus inserted into a separated pair of C-terminal helices, with some helical unfolding of the terminal chains in the striated muscle peptides. These overlap complexes are distinctly curved, much more so than elsewhere along the superhelical tropomyosin cable. To verify whether the non-native protein adducts (needed to stabilize the coiled-coil chimeras) perturb the overlap, we carried out Molecular Dynamics simulations of head-to-tail structures having only native tropomyosin sequences. We observe that the splayed chains all refold and become helical. Significantly, the curvature of both the smooth and the striated muscle overlap domain is reduced and becomes comparable to that of the rest of the tropomyosin cable. Moreover, the measured flexibility across the junction is small. This and the reduced curvature ensure that the super-helical cable matches the contours of F-actin without manifesting localized kinking and excessive flexibility, thus enabling the high degree of cooperativity in the regulation of myosin accessibility to actin filaments.

Periodicities designed in the tropomyosin sequence and structure define its functions

Tropomyosin is an actin binding protein that regulates actin filament dynamics and its interactions with actin binding proteins such as myosin, tropomodulin, formin, Arp2/3 and ADF-cofilin in most eukaryotic cells. Tropomyosin is the prototypical two-chained, α-helical coiled coil protein that associates end-to-end and binds to both sides of the actin filament. Each tropomyosin molecule spans four to seven actin monomers in the filament, depending on the size of the tropomyosin. Tropomyosins have a periodic heptad repeat sequence that is characteristic of coiled coil proteins as well as additional periodicities required for its interaction with the actin filament, where each periodic repeat interacts with one actin molecule. This review addresses the role of periodic features of the Tm molecule in carrying out its universal functions of binding to the actin filament and its regulation and the specific features that may determine the isoform specificity of tropomyosins.

Comprehensive Proteomics Approach in Characterizing and Quantifying Allergenic Proteins from Northern Shrimp: Toward Better Occupational Asthma Prevention

Occupational asthma is a major chronic health dilemma among workers involved in the seafood industry. Several proteins notoriously known to cause asthma have been reported in different seafood. This work involves the application of an allergenomics strategy to study the most potent allergens of northern shrimp. The proteins were extracted from shrimp tissue and profiled by gel electrophoresis. Allergenic proteins were identified based on their reactivity to patient sera and were structurally identified using tandem mass spectrometry. Northern shrimp tropomyosin, arginine kinase, and sarcoplasmic calcium-binding protein were found to be the most significant allergens. Multiple proteolytic enzymes enabled 100% coverage of the sequence of shrimp tropomyosin by tandem mass specrometry. Only partial sequence coverage was obtained, however, for the shrimp allergen arginine kinase. Signature peptides, for both tropomyosin and arginine kinase, were assigned and synthesized for use in developing the multiple reaction monitoring tandem mass spectrometric method. Subsequently, air samples were collected from a shrimp processing plant and two aerosolized proteins quantified using tandem mass specrometry. Allergens were detected in all areas of the plant, reaching levels as high as 375 and 480 ng/m(3) for tropomyosine and arginine kinase, respectively. Tropomyosine is much more abundant than arginine kinase in shrimp tissues, so the high levels of arginine kinase suggest it is more easily aerosolized. The present study shows that mass spectrometric analysis is a sensitive and accurate tool in identifying and quantifying aerosolized allergens.

Push and Pull of Tropomyosin’s Opposite Effects on Myosin Attachment to Actin. A Chimeric Tropomyosin Host−Guest Study

Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently influence myosin. One such influence is inhibitory and is due to tropomyosin's statistically preferred positions on actin that sterically interfere with actin's strong attachment site for myosin. Contrastingly, tropomyosin's other influence is activating. It increases myosin's overall actin affinity ∼4-fold. Stoichiometric considerations cause this activating effect to equate to an ∼4(7)-fold effect of myosin on the actin affinity of tropomyosin. These positive, mutual, myosin-tropomyosin effects are absent if Saccharomyces cerevisiae tropomyosin replaces mammalian tropomyosin. To investigate these phenomena, chimeric tropomyosins were generated in which 38-residue muscle tropomyosin segments replaced a natural duplication within S. cerevisiae tropomyosin TPM1. Two such chimeric tropomyosins were sufficiently folded coiled coils to allow functional study. The two chimeras differed from TPM1 but in opposite ways. Consistent with steric interference, myosin greatly decreased the actin affinity of chimera 7, which contained muscle tropomyosin residues 228-265. On the other hand, myosin S1 increased by an order of magnitude the actin affinity of chimera 3, which contained muscle tropomyosin residues 74-111. Similarly, myosin S1-ADP binding to actin was strengthened 2-fold by substitution of chimera 3 tropomyosin for wild-type TPM1. Thus, a yeast tropomyosin was induced to mimic the activating behavior of mammalian tropomyosin by inserting a mammalian tropomyosin sequence. The data were not consistent with direct tropomyosin-myosin binding. Rather, they suggest an allosteric mechanism, in which myosin and tropomyosin share an effect on the actin filament.

Electron Microscopy and Persistence Length Analysis of Semi-Rigid Smooth Muscle Tropomyosin Strands

The structural mechanics of tropomyosin are essential determinants of its affinity and positioning on F-actin. Thus, tissue-specific differences among tropomyosin isoforms may influence both access of actin-binding proteins along the actin filaments and the cooperativity of actin-myosin interactions. Here, 40 nm long smooth and striated muscle tropomyosin molecules were rotary-shadowed and compared by means of electron microscopy. Electron microscopy shows that striated muscle tropomyosin primarily consists of single molecules or paired molecules linked end-to-end. In contrast, smooth muscle tropomyosin is more a mixture of varying-length chains of end-to-end polymers. Both isoforms are characterized by gradually bending molecular contours that lack obvious signs of kinking. The flexural stiffness of the tropomyosins was quantified and evaluated. The persistence lengths along the shaft of rotary-shadowed smooth and striated muscle tropomyosin molecules are equivalent to each other (approximately 100 nm) and to values obtained from molecular-dynamics simulations of the tropomyosins; however, the persistence length surrounding the end-to-end linkage is almost twofold higher for smooth compared to cardiac muscle tropomyosin. The tendency of smooth muscle tropomyosin to form semi-rigid polymers with continuous and undampened rigidity may compensate for the lack of troponin-based structural support in smooth muscles and ensure positional fidelity on smooth muscle thin filaments.

Critical interactions in the stability control region of tropomyosin

Our laboratory has recently described a stability control region in the two-stranded alpha-helical coiled-coil alpha-tropomyosin that accounts for overall protein stability but is not required for folding (Hodges et al., 2009). We have used a synthetic peptide approach to investigate three stability control sites within the stability control region (residues 97-118). Two of the sites, electrostatic cluster 1 (97-104, EELDRAQE) and electrostatic cluster 2 (112-118, KLEEAEK), feature sequences with unusually high charge density and the potential to form multiple intrachain and interchain salt bridges (ionic attractions). A third site (105-111, RLATALQ) features an e position Leu residue, an arrangement known previously to enhance coiled-coil stability modestly. A native peptide and seven peptide analogs of the tropomyosin sequence 85-119 were prepared by Fmoc solid-phase peptide synthesis. Thermal stability measurements by circular dichroism (CD) spectroscopy revealed the following T(m) values for the native peptide and three key analogs: 52.9 degrees C (Native), 46.0 degrees C (R101A), 45.3 degrees C (K112A/K118A), and 27.9 degrees C (L110A). The corresponding DeltaT(m) values for the analogs, relative to the native peptide, are -6.9 degrees C, -7.6 degrees C, and -25.0 degrees C, respectively. The dramatic contribution to stability made by L110e is three times greater than the contribution of either electrostatic cluster 1 or 2, likely resulting from a novel hydrophobic interaction not previously observed. These thermal stability results were corroborated by temperature profiling analyses using reversed-phase high-performance liquid chromatography (RP-HPLC). We believe that the combined contributions of the interactions within the three stability control sites are responsible for the effect of the stability control region in tropomyosin, with the Leu110e contribution being most critical.

What makes tropomyosin an actin binding protein? A perspective

Tropomyosin is a two-chained alpha-helical coiled coil that binds along the length of the actin filament and regulates its function. The paper addresses the question of how a "simple" coiled-coil sequence encodes the information for binding and regulating the actin filament, its universal target. Determination of the tropomyosin sequence confirmed Crick's predicted heptapeptide repeat of hydrophobic interface residues and revealed additional features that have been shown to be important for its function: a 7-fold periodicity predicted to correspond to actin binding sites and interruptions of the canonical interface with destabilizing residues, such as Ala. Evidence from published work is summarized, leading to the proposal of a paradigm that binding of tropomyosin to the actin filament requires local instability as well as regions of flexibility. The flexibility derives from bends and local unfolding at regions with a destabilized coiled-coil interface, as well as from the dynamic end-to-end complex. The features are required for tropomyosin to assume the form of the helical actin filament, and to bind to actin monomers along its length. The requirement of instability/flexibility for binding may be generalized to the binding of other coiled coils to their targets.

CLONING,EXPRESSION AND IMMUNOCHARACTERIZATION OF ALLERGEN TROPOMYOSIN IN CRAB

Allergic disease is regarded as one of three diseases that need prevention and cure in the 21st century.Food hypersensitivity is a normal allergic disease,which can provokes dizziness,headache,chest distress,itch of skin and so on,so it is necessary to study the food allergens,especially seafood allergens.This study was undertaken to clone,express and immunocharacterize the allergen tropomyosin from crab(Portunus sanguinolentus).Degenerate primers were designed according to the conserved sequence of tropomyosin by means of bioinformatics and molecular biological methods.The RT-PCR was applied to clone the full-length allergen genes from crab and the sequences were analyzed.The specific primers were designed.The ORF of tropomyosin of crab was subcloned into the expression vector pET-28a.Expression of the recombinant crab tropomyosin was carried out in Escherichia coli BL21(DE3) and the purification of the recombinant protein was performed via affinity chromatography with Ni2+ coupled to sepharose.IgE reactivity of recombinant crab tropomyosin was investigated by Western-blot.After SDS-PAGE,the purified recombinant crab tropomyosin was digested by trypsin and analyzed by MALDI-TOF-MS.A novel full-length cDNA clone was obtained,which includes an open reading frame coding for 285 amino acids.The predicted amino acid sequences of the tropomyosin showed that its pI was approximately 4.71 and theoretical molecular weight was approximately 32.8kD.Sequence analysis showed that this gene shared high identities(80%)with allergen tropomyosin from many crustaceans and other invertebrates,such as 97%,92% and 83% identity with Chaf 1(Q9N2R3) from Charybdis feriatus,Mete 1(Q25456) from Metapenaeus ensis and Pera 7(Q9UB83) from Periplaneta Americana,respectively.The deduced protein was therefore regarded as crab allergen and named as Pors 1(EMBL/GenBank database entry No.EF143836).After over expressed in Escherichia coli BL21(DE3),the recombinant protein was purified through affinity chromatography with Ni2+ coupled to sepharose.Western-blot analysis showed that the recombinant allergen had IgE-reactivity with sera from crab allergic patients.The result of MALDI-TOF-MS indicated that this recombinant allergen was indeed the crab tropomyosin.In short,the crab tropomyosin was cloned and expressed successfully in BL21(DE3),which had reactivity with sIgE.All of these studies will be used as a base for further study on structure and function of crab allergens,also for the specific diagnosis and immunotherapy in crab hypersensitivity disease.

Allergen motifs and the prediction of allergenicity

We have recently shown that the majority of allergens can be represented by allergen motifs. This observation prompted us to experimentally investigate the synthesized peptides corresponding to the in silico motifs with regard to potential IgE binding and cross-reactions with allergens. Two motifs were selected as examples to conduct in vitro studies. From the first motif, derived from allergenic MnSOD sequences, the motif stretch of the allergen Asp f 6 was selected and synthesized as a peptide (MnSOD Mot). The corresponding full-length MnSOD was also expressed in Escherichia coli and both were compared for IgE reactivity with sera of patients reacting to the MnSOD of Aspergillus fumigatus or Malassezia sympodialis. For the second motif, the invertebrate tropomyosin sequences were aligned and a motif consensus sequence was expressed as a recombinant protein (Trop Mot). The IgE reactivity of Trop Mot was analyzed in ELISA and compared to that of recombinant tropomyosin from the shrimp Penaeus aztecus (rPen a 1) in ImmunoCAP. MnSOD Mot was weakly recognized by some of the tested sera, suggesting that the IgE binding epitopes of a multimeric globular protein such as MnSOD cannot be fully represented by a motif peptide. In contrast, the motif Trop Mot showed the same IgE reactivity as shrimp full-length tropomyosin, indicating that the major allergenic reactivity of a repetitive structure such as tropomyosin can be covered by a motif peptide. Our results suggest that the motif-generating algorithm may be used for identifying major IgE binding structures of coiled-coil proteins.

The structural role of high molecular weight tropomyosins in dipteran indirect flight muscle and the effect of phosphorylation

In Drosophila melanogaster two high molecular weight tropomyosin isoforms, historically named heavy troponins (TnH-33 and TnH-34), are encoded by the Tm1 tropomyosin gene. They are specifically expressed in the indirect flight muscles (IFM). Their N-termini are conventional and complete tropomyosin sequences, but their C-termini consist of different IFM-specific domains that are rich in proline, alanine, glycine and glutamate. The evidence indicates that in Diptera these IFM-specific isoforms are conserved and are not troponins, but heavy tropomyosins (TmH). We report here that they are post-translationally modified by several phosphorylations in their C-termini in mature flies, but not in recently emerged flies that are incapable of flight. From stoichiometric measurements of thin filament proteins and interactions of the TmH isoforms with the standard Drosophila IFM tropomyosin isoform (protein 129), we propose that the TmH N-termini are integrated into the thin filament structural unit as tropomyosin dimers. The phosphorylated C-termini remain unlocated and may be important in IFM stretch-activation. Comparison of the Tm1 and Tm2 gene sequences shows a complete conservation of gene organisation in other Drosophilidae, such as Drosophila pseudoobscura, while in Anopheles gambiae only one exon encodes a single C-terminal domain, though overall gene organization is maintained. Interestingly, in Apis mellifera (hymenopteran), while most of the Tm1 and Tm2 gene features are conserved, the gene lacks any C-terminal exons. Instead these sequences are found at the 3' end of the troponin I gene. In this insect order, as in Lethocerus (hemipteran), the original designation of troponin H (TnH) should be retained. We discuss whether the insertion of the IFM-specific pro-ala-gly-glu-rich domain into the tropomyosin or troponin I genes in different insect orders may be related to proposals that the IFM stretch activation mechanism has evolved independently several times in higher insects.

Identification of differentially expressed genes in primary varicose veins

A number of changes in protein expression have been described in primary varicose veins, but the altered gene expressions in this disease are unknown. The aim of this study was to identify differentially expressed genes in primary varicose veins. Total RNAs were isolated from two groups of greater saphenous veins (four primary varicose veins and three normal) and then were reverse transcribed into cDNAs. We used the differential display reverse transcription-polymerase chain reaction technique to screen the differences in the mRNA expression profiles of the groups. We found that three cDNAs showed differences in expression patterns between normal and diseased saphenous veins. The cDNAs are prominently expressed only in patients with varicose veins. We identified that the cDNAs had significant similarities to the L1M4 repeat sequence of clone RP11-57L9, clone RP11-299H13, and Alu repetitive sequence of human tropomyosin 4 mRNA. Our results suggest that the screened cDNA clones are useful disease markers in the genetic diagnosis of primary varicose vein and that the L1 and Alu elements possibly participated in the development of primary varicose veins through their expression patterns in genes encoded with structural proteins, such as collagen, elastin, and tropomyosin. Further studies are required to elucidate the potential relationship between repeat sequences and primary varicose veins.