Native Tropomyosin Research Articles

Protective immunity induced by vaccination with Onchocerca volvulus tropomyosin in rodents.

A cDNA clone of Onchocerca volvulus, designated MOv14, and encoding 136 amino-acid residues from the C-terminus of O. volvulus tropomyosin, was evaluated as a protective immunogen in two complimentary rodent models of onchocerciasis. Vaccination of BALB/c mice with the recombinant fusion of MOv14 coupled to Maltose-Binding Protein (MBP) induced significant reductions (48-62%) in the recovery of Onchocerca lienalis microfilariae from the skin, compared to control groups immunized with MBP alone. The predominant antibody response generated to MOv14 by vaccination was of IgG1. Following a similar vaccination protocol in Mongolian jirds, two independent experiments demonstrated that 16 weeks after infection with Acanthocheilonema viteae there was a 46% reduction in the recovery of adult worms in vaccinated animals compared to control groups. Antibodies generated by vaccination recognized a product released during culture of A. viteae infective larvae which migrated at a distinct molecular mass from native tropomyosin from somatic tissues.

Immunochemical characterization of recombinant and native tropomyosins as a new allergen from the house dust mite, Dermatophagoides farinae

Background: Two predominant mite species, Dermatophagoides farinae and Dermatophagoides pteronyssinus, are responsible for the immediate hypersensitivity reaction. Although a large number of antigens and allergens was detected in a whole mite culture extract, only the major allergens of Der I and II groups have been extensively studied. Much information on other important allergens remains to be accumulated. Methods: A new allergen complementary DNA (cDNA) clone was isolated from a D. farinae protein cDNA library. The expression product of the cDNA and native tropomyosin from D. farinae were purified, characterized, and evaluated. Results: An amino acid sequence deduced from the cDNA clone revealed significant homology with tropomyosins conserved in a wide range of animals. The amino acid sequences of two fragments obtained by degradation of the purified protein with cyanogen bromide coincided completely with the corresponding portions of the deduced amino acid sequence. The native tropomyosin reacted with specific IgE in the 31 sera tested at a high frequency (80.6%), comparable to that of Der f I (90.3%) and Der f II (74.2%). Conclusions: The cloned cDNA encodes tropomyosin. The high binding response of specific IgE antibodies to recombinant and native tropomyosins demonstrates that mite tropomyosin is an additional important allergen in house dust mite. ( J Allergy Clin Immunol 1995;96:74-83.)

Reannealing of beta-actinin-severed actin filaments by troponin and tropomyosin.

During a survey of actin pointed end-capping protein in salt extracts of rabbit skeletal muscle, it was found that native tropomyosin (troponin-tropomyosin complex) inhibited the seed activity of beta-actinin-capped actin filaments for actin polymerization. It turned out that beta-actinin-capped actin fragments reannealed to form long filaments resulting in the decrease in seed numbers in the presence of troponin and tropomyosin. It appears that the reannealing of actin filaments was due to release of beta-actinin from the actin filaments by troponin and tropomyosin. Either troponin or tropomyosin alone was not effective at all. Addition of an excess amount of beta-actinin did not prevent the reannealing of beta-actinin-capped actin filaments in the presence of troponin and tropomyosin.

Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins

The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSM beta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSM beta) and a mutant (rCSM beta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSM beta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 x 10(6) and 2.78 x 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSM beta) appeared to have a comparable actin-binding affinity to that of gel-purified CSM beta homodimer (1.25 x 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 x 10(7) M-1). The amino-terminal deletion mutant rCSM beta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSM beta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSM beta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSM beta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSM beta in the actin binding and the caldesmon enhancement.

Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation.

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).

Smooth muscle tropomyosin coiled-coil dimers. Subunit composition, assembly, and end-to-end interaction.

Subunits of gizzard smooth muscle tropomyosin, dissociated by guanidinium chloride and reassociated by high salt dialysis, form a 1:1 mixture of the beta beta and gamma gamma homodimers (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The homodimers have now been separated by anion-exchange chromatography and native gel electrophoresis, enabling us to show that the native protein is composed of more than 90% heterodimer. The in vitro equilibrium distribution of heterodimer and homodimers, at close to physiological temperature and ionic conditions, was calculated from thermal unfolding profiles of separated homodimers and heterodimer, as monitored by circular dichroism. The results, for an equal proportion of beta and gamma chains, indicate a predominant formation of heterodimer via chain dissociation and chain exchange, although the proportion of heterodimer was much less than the 90-100% found in the native protein. However, the proportion of heterodimer for actin-bound tropomyosin, determined by analyzing tropomyosin sedimented with actin, was greater than 90%, which may provide a model for assembly in vivo. The end-to-end interactions of the homodimers are about the same but are much less than that of the native heterodimer, as determined by viscometry. The greater end-to-end interaction of heterodimers may lead to stronger binding to actin compared to homodimers and thus would further shift the equilibrium between heterodimer and homodimers toward heterodimer and possibly account for the almost exclusive population of heterodimer in the presence of actin. The greater end-to-end interaction of the heterodimer may also provide a functional advantage for its preferred assembly. This study also shows that the two-step thermal unfolding of the homodimer mixture is due to the formation of heterodimer via an intermediate which is a new type of tropomyosin species which forms a gel in low salt. This tropomyosin is also present in small amounts in native tropomyosin preparations.

Purification and characterization of recombinant tropomyosins

The cloning of a cDNA coding for the skeletal human β-tropomyosin in the bacterial expression vector pKK233-2 is reported. Deletion mutants were also constructed. pCF-T1088 was obtained by elimination of exon 9 and pCF-T1089 was built by deleting 2/3 of the first exon. The recombinant tropomyosins were synthesized in E. coli after induction by IPTG. The mutant proteins were characterized by western blot using antibodies raised against native tropomyosin. The amount of the human protein synthesized in E. coli varies with each mutant, suggesting the involvement of the structure of the protein or of the mRNA on the synthesis or the stability of the recombinant protein. After precipitation of most of the bacterial proteins at 100°C, purification was achieved by high-performance liquid chromatography (HPLC) using TSK-DEAE, hydroxyapatite and reversed-phase columns. The chromatographic behaviour of the mutants were compared. Characterization of the mutated tropomyosins was achieved by tryptic digestion and analysis of the peptide composition by reversed-phase HPLC. A computer program for predicting the retention times of the peptides generated was written. It is shown that it is possible to identify the mutations solely by comparing the chromatogram of the tryptic digest with the profile obtained by computer simulation.

Binding Mode of Cytochalasin B to F-Actin Is Altered by Lateral Binding of Regulatory Proteins

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering

Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 μg/ml to enable free movement of neighboring filaments and yet give a reliable signal. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments.

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.

Regulation of myosin sliding alongChara actin bundles by native skeletal muscle tropomyosin

Native tropomyosin from rabbit skeletal muscle introduced by intracellular perfusion intoChara cells inhibited the cytoplasmic streaming irrespective of the Ca2+ concentration. To find the action site of native tropomyosin inChara, the cytoplasmic streaming was reconstituted by introducing isolated endoplasm into actin donorChara cells from which native endoplasm had been removed. The reconstituted streaming was inhibited by pretreatment of the actin donor cells with native tropomyosin but not by that of the endoplasm, suggesting that the native tropomyosin inhibited the cytoplasmic streaming by binding toChara actin bundles. Staining of the actin bundles with FITC-labeled native tropomyosin also showed that the native tropomyosin could bind to the actin bundles. Streaming reconstituted fromChara actin bundles and skeletal muscle myosin was insensitive to Ca2+, but became sensitive on application of the native tropomyosin.

Native chicken gizzard tropomyosin is predominantly a beta gamma-heterodimer.

Unmodified chicken gizzard tropomyosin (TM) has been fractionated into its two major isoforms beta and gamma, by chromatofocussing in the presence of 9 M urea and dithiothrieitol. Treatment of the native protein with several bifunctional N-hydroxysuccinimide esters gave the beta gamma-heterodimer as the major cross-linked product. A comparison of the thermal transition profiles of the two homodimers and of the native unfractionated TM also indicated the predominance of the beta gamma-heterodimer in the native protein. This conclusion is consistent with the absence of excimer fluorescence in pyrene-labeled gizzard TM and the relative resistance of the molecule to intramolecular disulfide formation (Lehrer, S.S., Betteridge, D.R., Graceffa, P., Wong, S., and Seidel, J. C. (1984) Biochemistry 23, 1591-1595) since the single cysteines on each of the two isoforms are widely separated. We conclude that further experimental evidence is required to assess the possibility that the gizzard TM is more rigid in its conformation than are those of the skeletal and cardiac proteins.

Enzymatic properties of myosin from ascidian body-wall smooth muscle

1. 1. Myosin from ascidian body-wall smooth muscle was purified, and 2. 2. The ATPase activity of the myosin activated by actin was insensitive to Ca 2+. 3. 3. However, in the presence of ascidian native tropomyosin (a complex of tropomyosin and troponin), the ATPase activity was Ca 2+-sensitive. 4. 4. The activity was suppressed by removing free Ca 2+ from the reaction mixture with EGTA, and the suppression was released by this ion. 5. 5. The properties of myosin from ascidian were very similar to those of the protein from rabbit skeletal muscle.

Factors influencing interaction of phosphorylated and dephosphorylated myosin with actin

The influence of various factors on the interaction of phosphorylated and dephosphorylated myosin with action was examined. It was found that the difference between the values of specific activity of the two myosin forms of actin-stimulated Mg 2+-ATPase is affected by changes in KCl, MgATP and acting concentration. The effect of increased pH on the differences in the rate of ATP hydrolysis by actomyosin containing phosphorylated myosin as compared with that of the dephosphorylated one, observed in the presence of EGTA, is abolished by addition of Ca 2+. Tropomyosin strongly inhibits the actin-stimulated Mg 2+-ATPase of phosphorylated myosin (by about 60%). The tropomyosin-troponin complex and native tropomyosin lowered the rate of ATP hydrolysis by actomyosin containing both phosphorylated and dephosphorylated myosin by about of 60% of the value obtained in the absence of those proteins. These results indicate that the change of negative charge on the myosin head due to phosphorylation and dephosphorylation of myosin light chains modulates the actin-myosin interaction at different steps of the ATP hydrolysis cycle. Phosphorylation of myosin seems to be a factor decreasing the rate of ATP hydrolysis by actomyosin under physiological conditions.

Calcium binding of arterial tropomyosin: involvement in the thin filament regulation of smooth muscle.

Bovine aortic tropomyosin has been isolated by DEAE-Sepharose chromatography following isoelectric precipitation and ammonium sulfate fractionation. A single polypeptide [Mr 36 000 on a sodium dodecyl sulfate (SDS)-polyacrylamide gel] was obtained under different electrophoretic conditions. The amino acid composition of bovine tropomyosin was very similar to that of rabbit skeletal muscle; the amino-terminal residue is blocked. The molecular weight of the native tropomyosin (76 000), which is twice that calculated from the SDS-polyacrylamide gel, suggests that the molecule is a dimer. The diffusion coefficient of 3.4 X 10(-7) cm2 s-1 and the frictional coefficient of 1.7 indicate that the molecule is asymmetric. Comparative high-pressure liquid chromatography peptide mapping of rabbit skeletal and bovine aortic tropomyosins shows primary structure variation. Bovine aortic tropomyosin binds calcium under physiological conditions of pH and ionic strength (22 mol of Ca2+/mol of tropomyosin with a Kd of 1.4 mM). Such a property is not shared by skeletal tropomyosin. In low Mg2+ concentration, both skeletal and aortic actin activations of the skeletal myosin ATPase activity are calcium independent. Addition of aortic tropomyosin to a hybrid actomyosin (aortic actin, skeletal myosin) yields an enhancement of the actin activation of the myosin ATPase activity, but the addition of skeletal tropomyosin yields a decrease of this activity. However, both the enhancement and decrease are calcium dependent. Addition of skeletal or aortic tropomyosin to an actomyosin system, where both actin and myosin come from skeletal muscle, yields only an enhancement of the actin activation of the myosin ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine modification of myosin from chicken gizzard.

Previously, we (Suzuki et al. (1978) J. Biochem. 84, 1529) reported that the sedimentation constant of chicken gizzard myosin in the presence of ATP was approximately 10S in 0.15 M or 0.2 M KCl and approximately 6S in 0.3 M or higher concentrations of KCl. The 10S-myosin and 6S-myosin were considerably different in conformation from each other. I now report the finding that the transformation of 6S-myosin to the 10S conformation results in a drastic change in the reactivity of thiol groups of gizzard myosin with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (abbreviated as IAEDANS). The so-called SH1-type thiol groups (Sekine et al. (1962) J. Biol. Chem. 237, 2769) were present on 68 kilodalton fragments (produced by tryptic digestion) of gizzard myosin. The reactivity of the thiol groups with IAEDANS was greatly decreased by the 6S to 10S transformation of gizzard myosin molecules. Two other findings were obtained. Blocking the SH1-type thiol groups made the Mg-ATPase activities (in the presence of gizzard native tropomyosin) of gizzard myosin and of acto-gizzard myosin insensitive to calcium and to phosphorylation of regulatory light chains, although calcium-dependent phosphorylation of the IAEDANS-modified myosin could still occur. It also made gizzard myosin filaments resistant to the disassembly action of ATP.

Ca2+ regulation of myosin sliding along chara actin bundles mediated by native tropomyosin.

Ca2+ regulation of myosin sliding along chara actin bundles mediated by native tropomyosin.

The contractile activity of leukocyte contractile protein

Contractile activity of myosin-B from porcine leukocytes was regulated by Ca2+, which was associated with the level of phosphorylation of its myosin light chain. In another series of experiment, it was found that the myosin-B lost its contractility by washing with 1 mM NaHCO3. However, the washed myosin-B restored its Ca2+-sensitive contractility by the addition of not only a fraction prepared from the NaHCO3-wash of the myosin-B, but also “native tropomyosin (NT) ” which was the regulatory protein fraction from arterial smooth muscle. From the above results, its was considered that the mechanism of Ca2+-sensitive contractility of leukocyte-myosin-B was analogous to that of arterial contractile protein. Some preparations of leukocyte-myosin-B showed the same properties as the washed myosin-B in the Ca2+-sensitive contractile activity. In the myosin B, the Mg2+-ATPase activity was regulated by Ca2+ through “NT” fraction from arterial smooth muscle. On the other hand, leukocyte-myosin and arterial tropomyosin (TM) were purified from leukocyte-myosin B and arterial “NT”, respectively. In a synthetic actomyosin consisting of skeletal actin and leukocyte-myosin, arterial NT enhanced the actinactivated Mg2+-ATPase activity of leukocyte-myosin. The enhancement was much higher in the presence of Ca2+ and associated with an increase in the level of phosphorylation of myosin light chain. The arterial TM enhanced also the Mg2+-ATPase activity of the synthetic actomyosin, but its enhancement was independent of Ca2+. The enhancement was lower than that of NT and was not associated with an increase in the phosphorylation of myosin light chain. These results indicate that leukocyte contractile protein is essentially regulated by the level of Ca2+-dependent myosin light chain phosphorylation, but its contractility is augumented by TM associated with actin, independent of Ca2+

Loss of Ca2+-dependent regulation in glycerinated skeletal muscle contraction.

Glycerinated muscle fiber from rabbit psoas muscle often lost Ca2+-dependent regulation of its contraction with long-term extraction in a 50% glycerol solution containing 5 mM EGTA at -20 degrees C, designated as Ca2+-insensitive muscle fiber (CaIS-fiber). About 30 or 40% of glycerinated muscle fibers were CaIS-fibers after 1 to 3 months in the glycerol solution. We investigated the cause of the loss of Ca2+-sensitivity of the glycerinated muscle fiber by tension mechanogram and SDS polyacrylamide gel electrophoresis. This natural CaIS-fiber showed a new band of 30K daltons peptide on SDS gels. On the other hand, Ca2+-sensitive fiber (CaS-fiber) changed to CaIS-fiber by trypsin digestion for 40 sec. The tryptic CaIS-fiber had no troponin C and 30K daltons peptide bands in the electrophoretograms. Incubating with 2 mM CaCl2 for 40 hr at 25 degrees C, CaS-fiber changed easily to CaIS-fiber which had 30K daltons peptide and faint troponin T and I bands, as in natural CaIS-fiber. All CaIS-fibers could recover their Ca2+-dependent regulation by incubating with native tropomyosin from rabbit skeletal muscle for 2 days at 4 degrees C. These results indicate that the loss of Ca2+-dependent regulation of glycerinated muscle fiber is due to degradation of regulatory protein system by endogenous Ca2+-activated proteolytic enzymes.

Crayfish myosin has no Ca2+-dependent regulation in actomyosin.

1. Crayfish myosin B lost Ca2+-dependent regulation in superprecipitation upon addition of pure rabbit skeletal F-actin. 2. Actomyosin reconstituted from crayfish myosin and pure rabbit skeletal F-actin showed both Ca2+-dependent regulation in superprecipitation and Mg2+-ATPase activity upon addition of native tropomyosin prepared from crayfish or rabbit skeletal muscles. Also, superprecipitation of this actomyosin was induced by ITP without Ca2+-dependent regulation, as is the case in rabbit skeletal actomyosin. 3. Actomyosin reconstituted from crayfish native thin filament and crayfish or rabbit skeletal myosins showed Ca2+-dependent regulation. 4. These findings indicate that crayfish myosin is similar to rabbit skeletal myosin and different from chicken gizzard myosin in regulatory function.