Genomic structure of the sandworm, Perinereis vancaurica tetradentata, troponin C

Troponin C (TnC) superfamily genes essentially possess five introns, the positions of all but the fourth being highly conserved. The fourth intron is frequently absent from protostomian invertebrate genes, such as calmodulin or TnC. We previously proposed that the common ancestor of TnC superfamily genes never possessed an intron corresponding to today's fourth introns, and that members of the superfamily independently gained a fourth intron in the evolutionary pathway of each lineage. In the present study, we isolated the TnC cDNA from the sandworm, Perinereis vancaurica tetradentata and determined its genomic structure. Sandworm TnC appears to exist as a single copy gene consisting of six exons and five introns. The positions of the first, second, third and fifth introns are identical to other TnCs, but that of the fourth intron is unique. This is in good agreement with the above-mentioned scheme, i.e. the gain of the fourth intron of sandworm TnC might have occurred within the annelid lineage after annelida/mollusca divergence.     

Structural organization of lower marine nonvertebrate calmodulin genes.

The troponin C (TnC) superfamily genes generally possess five introns, and the positions where they are inserted are well conserved except for the fourth intron. Based on a structural comparison of TnC genes, we proposed that the common ancestor of TnC or TnC superfamily genes had no intron corresponding to the modern fourth intron, and therefore members of the superfamily have gained the fourth intron independently within each lineage. Here, we cloned calmodulin (CaM, one of the members of the TnC superfamily) cDNAs from two lower marine nonvertebrates, the sea anemone, Metridium senile, belonging to the Cnidaria, and the sponge, Halichondria okadai, belonging to the Porifera, and also determined their genomic organization. Chordate CaM genes generally possess five introns, but neither sea anemone nor sponge CaM has anything corresponding to the fourth intron of chordate CaMs, suggesting that the early metazoan CaM must have had only four introns. The modern fourth intron of chordate CaMs was acquired within the chordate lineage after nonvertebrate/chordate divergence. This notion concurs with our proposal explaining the evolution of the TnC superfamily genes.     

Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).     

Infrared spectroscopic study of the binding of divalent cations to Akazara scallop troponin C: the effect of the methylene side chain of glutamate residue

Akazara scallop striated adductor muscle troponin C (TnC) binds only one Ca2+ because the three EF-hand motifs are short of critical residues for the coordination of Ca2+. Fourier-transform infrared spectroscopy was applied to study coordination structures of M2+ (= Mg2+, Ca2+, Sr2+, and Ba2+) bound in an Akazara scallop TnC mutant (E142D) and the wild-type TnC C-lobe in D2O solution. The region of the COO- antisymmetric stretch provides information regarding the coordination modes of a COO- group to a metal ion. The side chain COO- group of Asp142 did not bind to Ca2+ in the bidentate coordination mode, suggesting that the absence of a methylene group is critical for the Ca2+ coordination structure of Akazara scallop TnC (Nara et al., Vib Spect 2006, 42, 188-191). The present study has shown that the absence of a methylene group is not compensated for by a larger metal ion such as Sr2+ or Ba2+. CD spectra showed that the secondary structures are conserved between M2+-free (apo), Mg2+-loaded, Ca2+-loaded, Sr2+-loaded, and Ba2+-loaded states, which was consistent with the results estimated from their amide I band patterns. The metal-ligand interaction at position 12 of site IV is discussed in comparison with the coordination mode of the side chain COO- group of the wild-type TnC C-lobe.     

Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle

Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca²⁺ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca²⁺-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca²⁺-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca²⁺-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca²⁺-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca²⁺-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca²⁺ signal to TnI in Tn ternary complex.     

Developmentally regulated troponin C mRNAs of chicken skeletal muscle.

Fast and slow/cardiac troponin C (TnC) are the two different isoforms of TnC. Expression of these isoforms is developmentally regulated in vertebrate skeletal muscle. Therefore, in our studies, the pattern of their expression was analyzed by determining the steady-state levels of both TnC mRNAs. It was also examined if mRNAs for both isoforms of TnC were efficiently translated during chicken skeletal muscle development. We have used different methods to determine the steady-state levels of TnC mRNAs. First, probes specific for the fast and slow TnC mRNAs were developed using a 390 base pair (bp) and a 255 bp long fragment, of the full-length chicken fast and slow TnC cDNA clones, respectively. Our analyses using RNA-blot technique showed that fast TnC mRNA was the predominant isoform in embryonic chicken skeletal muscle. Following hatching, a significant amount of slow TnC mRNA began to accumulate in the skeletal (pectoralis) muscle. At 43 weeks posthatching, the slow TnC mRNA was nearly as abundant as the fast isoform. Furthermore, a majority of both slow and fast TnC mRNAs was found to be translationally active. A second method allowed a more reliable measure of the relative abundance of slow and fast TnC mRNAs in chicken skeletal muscle. We used a common highly conserved 18-nucleotide-long sequence towards the 5'-end of these mRNAs to perform primer extension analysis of both mRNAs in a single reaction. The result of these analyses confirmed the predominance of fast TnC mRNA in the embryonic skeletal muscle, while significant accumulation of slow TnC mRNA was observed in chicken breast (pectoralis) muscle following hatching. In addition to primer extension analysis, polymerase chain reaction was used to amplify the fast and slow TnC mRNAs from cardiac and skeletal muscle. Analysis of the amplified products demonstrated the presence of significant amounts of slow TnC mRNA in the adult skeletal muscle.     

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.     

Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Slow troponin C is present in both muscle and nonmuscle cells.

A common primer was used for synthesis of cDNAs from both chicken fast and slow troponin C (TnC) mRNAs. Synthesis of double-stranded cDNAs and their amplification by polymerase chain reaction gave specific products corresponding to these mRNAs. This method was used for determining the presence of TnC mRNAs in various tissues. Our results show that while the fast TnC mRNA is expressed only in the muscle cells, slow TnC mRNA is expressed in a number of nonmuscle cells. Not all nonmuscle cells, however, express slow TnC mRNA. Liver and brain tissues showed the presence of high levels of this mRNA, while it was absent in chicken smooth muscle and embryonic skin. The slow TnC mRNA was very stable in cardiac muscle cells. It degrades with a half-life of approximately 94 h. The same mRNA was less stable in skeletal muscle and liver cells. The half-lives were found to be only between 13 and 16 h in these cells. Our results suggest that slow TnC mRNA may function as the nonmuscle isoform of this contractile protein. Since slow TnC mRNA is the only TnC isoform present in cardiac muscle, liver and brain, it is possible that besides its role in regulating contraction of striated muscle slow TnC can also function in processes other than muscle contraction.     

Electrophoretic and functional identification of two troponin C isoforms in toad skeletal muscle fibers

The differential sensitivity of frog twitch and slow-tonic fibers to Ca²⁺ and Sr²⁺ suggests that these two fiber types express different troponin C (TnC) isoforms. To date, only one TnC isoform from anurans (resembling the mammalian fast-twitch isoform) has been isolated and characterized. In this study, we examined the possibility that anuran striated muscle contains more than one TnC isoform. Toward this end, we determined the TnC isoform composition of 198 single fibers from the rectus abdominis of the cane toad (a mixed slow-tonic and twitch muscle) and of toad cardiac muscle using a method that enables the identification of TnC isoforms on the basis of the effect of Ca²⁺ on their electrophoretic mobility. The fibers were typed according to their myosin heavy chain (MHC) isoform composition. The data indicate that striated muscle of the cane toad contains two TnC isoforms, one of which (TnC-t) is present in all fibers displaying only twitch MHC isoforms and the other of which (TnC-T/c) is present in fibers displaying the tonic MHC isoform and in cardiac muscle. For a subpopulation of 15 fibers, the TnC isoform composition was also compared with Ca²⁺ and Sr²⁺ activation characteristics. Fibers containing the TnC-T/c isoform were 3-fold more sensitive to Ca²⁺, 40-fold more sensitive to Sr²⁺, and responded to a 4.6-fold broader range of [Ca²⁺] than did fibers containing the TnC-t isoform. The Ca²⁺ activation properties of toad fibers containing the TnC-T/c isoform appear to be consistent with the previously reported physiological characteristics of amphibian slow-tonic muscle fibers. myofibrillar proteins; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; alanine SDS-PAGE; hybrid fibers; Ca²⁺-binding proteins; single fiber; muscle protein polymorphism; fiber type     

Isolation of a cDNA encoding the motor domain of nonmuscle myosin which is specifically expressed in the mantle pallial cell layer of scallop (Patinopecten yessoensis)

It has been reported that catch and striated muscle myosin heavy chains of scallop are generated through alternative splicing from a single gene [Nyitray et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12686-12690]. They suggested that the catch muscle type myosin was expressed in various tissues of scallop, including the gonad, heart, foot, and mantle. However, there have been no reports of the primary structure of myosin from tissues other than the adductor muscles. In this study, we isolated a cDNA encoding the motor domain of myosin from the mantle tissue of scallop (Patinopecten yessoensis), and determined its nucleotide sequence. Sequence analysis revealed that mantle myosin exhibited 65% identity with Drosophila non muscle myosin, 60% with chicken gizzard smooth muscle myosin, and 44% with scallop striated muscle myosin. The mantle myosin has inserted sequences in the 27 kDa domain of the head region, and has a longer loop 1 structure than those of scallop striated and catch muscle myosins. Phylogenetic analysis suggested that the mantle myosin is classified as a smooth/nonmuscle type myosin. Western blot analysis with antibodies produced against the N-terminal region of the mantle myosin revealed that this myosin was specifically expressed in the mantle pallial cell layer consisting of nonmuscle cells. Our results show that mantle myosin is classified as a nonmuscle type myosin in scallop.     

The calcium-induced switch in the troponin complex probed by fluorescent mutants of troponin I.

The Ca2+-induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the troponin complex.     

Functional and evolutionary relationships of troponin C.

Striated muscle contraction is initiated when, following membrane depolarization, Ca(2+) binds to the low-affinity Ca(2+) binding sites of troponin C (TnC). The Ca(2+) activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle.     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

动物肌动蛋白基因中内含子的来源及存在意义的探讨

对动物界演化过程中肌动蛋白家族内含子插入位置分布的演化规律作了分析,并对相同插入位置的内含子序列按同亚型和不同亚型作了比较。结果得出:从整个肌动蛋白家族的外显子序列高度保守性推断整个肌动蛋白家族可能是从共同的祖先蛋白进化而来的;从同亚型肌动蛋白内含子序列的类似性随进化距离而变化,但在短进化距离的物种间,类似性都较高,不同亚型肌动蛋白内含子序列的类似性都较低,即使是同一物种(如人),类似性也远低于同亚型但进化距离较近的物种,由此可推断,同亚型肌动蛋白的内含子序列可能从共同祖先进化,不同亚型肌动蛋白的内含子序列从不同祖先进化,综上推断可导出内含子可能是在蛋白异化过程中获得的:还发现内含子在肌动蛋白家族编码基因中位置的分布随进化方向不同而逐步形成两种截然不同的模式,由此提出了内含子的位置分布与动物演化方向之间可能具有某种必然联系,为内含子的存在提出了某种依据。     

Functional role of Ca(2+)-binding site IV of scallop troponin C

Scallop troponin C (TnC) binds only one Ca(2+)/mol and the single Ca(2+)-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, H. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca(2+)-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca(2+)-regulation was investigated using a site-directed mutant with an inactivated site IV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and wild-type TnC (TnC(W)). Equilibrium dialysis using (45)Ca(2+) demonstrated that TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca(2+)/mol, but that TnC-ZEQ and TnC(N) bind virtually no Ca(2+). The UV difference spectra of TnC(W) and TnC(C) showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca(2+)-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addition, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl(2), unlike TnC-ZEQ and TnC(N). These results indicate that Ca(2+) binds only to site IV and that Ca(2+)-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N), were shown to form soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and TnC(W) conferred Ca(2+)-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca(2+)-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca(2+)-sensitivity. Thus, the major functions of scallop TnC, such as Ca(2+)-binding and interaction with TnI, are located in the C-terminal domain, however, the full Ca(2+)-regulatory function requires the presence of the N-terminal domain.     

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.     

Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C.

A conformational change accompanying Ca2+ binding to troponin C (TnC) constitutes the initial event in contractile regulation of vertebrate striated muscle. We replaced endogenous TnC in single skinned fibers from rabbit psoas muscle with a modified form of cardiac TnC (cTnC) which, unlike native cTnC, probably contains an intramolecular disulfide bond. We found that such activating TnC (aTnC) enables force generation and shortening in the absence of calcium. With aTnC, both force and shortening velocity were the same at pCa 9.2 and pCa 4.0. aTnc could not be extracted under conditions which resulted in extraction of endogenous TnC. Thus, aTnC provides a stable model for structural studies of a calcium binding protein in the active conformation as well as a useful tool for physiological studies on the primary and secondary effects of Ca2+ on the molecular kinetics of muscle contraction.