Sequences of complete cDNAs encoding four variants of chicken skeletal muscle troponin T

cDNAs containing the complete coding sequences of four isoforms of troponin T derived from 1-week-old chick skeletal muscle have been isolated and sequenced. While the 5' and 3' untranslated regions and most of the coding sequence were identical for each, dramatic differences were observed in the NH2-terminal region corresponding to amino acid residues 10-37 of rabbit skeletal troponin T. These sequence differences correspond to the alternatively spliced but not mutually exclusive exons 4 to 8 of the rat skeletal muscle troponin T gene. In addition, we observe a sequence corresponding to an extra exon or exons (between 5 and 6) present in the chicken skeletal muscle gene and not previously detected in the rat skeletal or chicken cardiac genes. This sequence of 63 nucleotides consists of an almost perfect repeat of 30 and 33 nucleotides and has previously been shown to be represented as a protein variant in chicken skeletal muscle. A difference is also present in one cDNA clone corresponding to the alternatively spliced (mutually exclusive) exons 16 and 17 of the rat gene. In the protein, this corresponds to a region implicated in the interaction of troponin T with troponin C, tropomyosin, and perhaps troponin I and F-actin.     

Amino acid sequence of bovine cardiac troponin I

Troponin I (TnI) is the inhibitory subunit of troponin, the thin filament regulatory complex which confers calcium sensitivity to striated muscle actomyosin ATPase activity. We have determined the amino acid sequence of TnI from adult bovine cardiac muscle. This protein is a single polypeptide chain of 211 amino acids with an acetylated amino terminus, a calculated molecular weight of 23,975, and a net charge of +17 at neutral pH. There was no evidence for heterogeneity of the sequence. Comparison with other available TnI sequences shows an amino-terminal extension of 27-33 residues which is present in cardiac but not skeletal TnI. The remainder of the polypeptide is common to both cardiac and skeletal TnI. In the amino-terminal half of the common polypeptide, only 29% of the residues are invariant in all sequences. The carboxyl-terminal half (residues 124-210) is much more highly conserved, with 66% invariant residues. Bovine cardiac TnI and rabbit cardiac TnI are very similar in sequence: only 12 of 26 residues are identical in the amino-terminal segments, but the remaining residues of the proteins are 97% identical.     

Homology in protein sequences expressed by correlation coefficients

Internal homologies in an amino acid sequence of a protein and in amino acid sequences of two different proteins are examined, using correlation coefficients calculated from the sequences when residues are replaced by various quantitative properties of the amino acids such as hydrophobicity. To improve the signal-noise ratio the average correlation coefficient is used to detect homology because the correlation depends on the property considered. In this way, any sequence repetition in a protein and the extent of the similarity and difference among proteins can be estimated quantitatively. The procedure was applied first to the sequences of proteins which have been assumed on other grounds to contain some internal sequence repetitions, α-tropomyosin from rabbit skeletal muscle, calmodulin from bovine brain, troponin C from skeletal and cardiac muscle, and then to the sequences of calcium binding proteins, calmodulin, troponin C, and L2 light chain of myosin. The results show that α-tropomyosin has a markedly periodic sequence at intervals of multiples of seven residues throughout the whole sequence, and calmodulin and skeletal troponin C contain two homologous sequences, the homology of troponin C being weaker than that of calmodulin. Candidates for the calcium binding regions of both troponin C, calmodulin, and L2 light chain are the homologous parts having a high average correlation coefficient (about 0·5) with respect to the sequences of the CD and EF hand regions of carp parvalbumin. The procedure may be a useful method for searching for homologous segments in amino acid sequences.     

A cardiac troponin T epitope conserved across phyla.

Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.     

Bovine cardiac troponin T: amino acid sequences of the two isoforms

Troponin T (TnT) is the tropomyosin-binding subunit of troponin, the thin filament regulatory complex that confers calcium sensitivity to striated muscle contraction and actomyosin ATPase activity. Bovine cardiac muscle contains two isoforms (TnT-1 and TnT-2) of TnT that differ in sequence near their amino termini. Thin filaments containing TnT-2 require less calcium to activate the MgATPase rate of myosin than do thin filaments containing TnT-1. Using whole troponin T purified from adult bovine cardiac muscle, we have determined the complete amino acid sequence of the larger, more abundant isoform TnT-1. We confirmed that sequence differences between TnT-1 and TnT-2 are confined to the amino-terminal regions and found that TnT-1 makes up approximately 75% of the total troponin T isolated. Partial sequencing of the separated isoforms showed that the difference between them is due solely to residues 15-19 (Glu-Ala-Ala-Glu-Glu) of TnT-1 being absent from TnT-2. The deleted segment may correspond to the product of exon 4 of the chicken cardiac TnT gene [Cooper, T.A., & Ordahl, C.P. (1985) J. Biol. Chem. 260, 11140-11148]. Exon 5, which is developmentally regulated in the chicken, is not expressed in either TnT-1 or TnT-2. TnT-1 contains 284 amino acid residues and has a Mr of 33,808, while TnT-2 contains 279 amino acid residues and has a Mr of 33,279. Bovine cardiac TnT contains the only known thiol group in any isolated TnT (Cys-39 of TnT-1, Cys-34 of TnT-2). Comparison of bovine, rabbit, and chicken cardiac TnT sequences shows near identity of the amino-terminal 13 amino acid residues (exons 2 and 3 of the chicken cardiac gene), many differences in the following 60 residues (exons 4-8), and great similarity in the C-terminal 230 residues (exons 9-18).     

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.     

Molecular polarity in tropomyosin-troponin T co-crystals.

New features of the structure and interactions of troponin T and tropomyosin have been revealed by electron microscopy of so-called double-diamond co-crystals. These co-crystals were formed using rabbit alpha2 tropomyosin complexed with troponin T from either skeletal or cardiac muscle, which have different lengths in the amino-terminal region, as well as a bacterially expressed skeletal muscle troponin T fragment of 190 residues that lacks the amino-terminal region. Differences in the images of the co-crystals have allowed us to establish the polarities of both the troponin T subunit and tropomyosin in the projected lattice. Moreover, in agreement with their sequences, the amino-terminal region of a bovine cardiac muscle troponin T isoform appears to be longer than that from the rabbit skeletal muscle troponin T isoform and to span more of the amino terminus of tropomyosin at the head-to-tail filament joints. Images of crystals tilted relative to the electron beam also reveal the supercoiling of the tropomyosin filaments in this lattice. Based on these results, a three-dimensional model of the double-diamond lattice has been constructed.     

Amino acid sequence of chicken gizzard beta-tropomyosin. Comparison of the chicken gizzard, rabbit skeletal, and equine platelet tropomyosins

Chicken gizzard beta-tropomyosin has the same chain length (284 residues) as other muscle tropomyosins, and is most closely related to the beta component of rabbit skeletal muscle. The majority of the amino acid substitutions are restricted to two regions of the structure, residues 185-216 and 258-284. The altered sequences at the COOH-terminal ends (residue 258-284) of the two gizzard components are very similar to each other and to those in platelet tropomyosin and can be correlated with the reduced affinity of interaction of all three tropomyosins with skeletal troponin T and its T1 fragment. The virtually identical NH2-terminal sequences of all four muscle tropomyosin chains indicates that the gizzard proteins' greater ability to polymerize head-to-tail is due to the sequence changes at its COOH terminus. On the other hand, the weaker head-to-tail aggregation of the platelet protein must be due to its NH2-terminal sequence alterations. Examination of the distribution of amino acids and the frequency of their substitution in the a to g positions of the repeating pseudoheptapeptide for all five tropomyosin sequences (four muscle and one platelet) emphasizes the importance of Glu residues at position e. Examination of those features of the muscle sequences implicated in the stabilization of their coiled-coil structures and in their interactions with F-actin suggest only marginal differences among them, with the possible exception of the chicken gizzard gamma component.     

Troponin from the myocardium and skeletal muscles: structure and properties

The literary and experimental data on the structure and properties of cardiac and skeletal muscle troponin are reviewed. The cation--binding sites of cardiac and skeletal muscle troponin C are distinguished by specificity; the sites localized in the C-terminal part of the protein molecule can bind both Ca2+ and Mg2+, whereas the sites localized at the N-end specifically bind Ca2+. The use of bifunctional reagents revealed a number of helical sites within the structure of cardiac troponin C (residues 84-92 and 150-158) and of skeletal muscle troponin C (residues 90-98 and 125-136). A comparison of experimental data with the results of an X-ray analysis testifies to the presence in the central part of the troponin C molecule of a long alpha-helical sequence responsible for troponin C interaction with the inhibiting peptide of troponin I. The efficiency of interaction of troponin components depends on Ca2+ concentration; the integrity of the overall troponin complex is mainly provided for by troponin C interaction with troponin I and by troponin I interaction with troponin T. The interaction between troponins T and C is relatively weak, especially in the case of cardiac troponin components. Both skeletal and cardiac muscles synthesize several troponin T isoforms differing in length and amino acid composition of N-terminal 40-60 member peptides. Troponin T isoforms can undergo phosphorylation by several protein kinases. The single site of troponin T which exists in a phosphorylated state in vivo (residue Ser-1) undergoes phosphorylation by specific protein kinase (troponin T kinase) related to casein kinases II. It was assumed that the phosphorylation of Ser-1 residue of troponin T as well as the synthesis of troponin T isoforms differing in the structure of the N-terminal peptide, provides for the regulation of interaction between two neighbouring tropomyosin molecules.     

Identification of a region of fast skeletal troponin T required for stabilization of the coiled-coil formation with troponin I

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.     

Determination of the complete amino acid sequence of bovine cardiac troponin C.

The amino acid sequence of bovine cardiac troponin C has been completely determined. The protein was cleaved by cyanogen bromide and the resulting peptides were isolated. All of the 161 residues of the protein could be accounted for in 12 cyanogen bromide peptides. Overlapping peptides were generated by tryptic digestion of citraconylated troponin C and isolation of the resulting five peptides. The primary structure of cardiac troponin C was elucidated by sequential manual Edman degradation of these peptides. It consists of four homologous regions, one of which probably has lost the ability to bind calcium ions. By comparing the amino acid sequence of cardiac troponin C with the sequence of skeletal troponin C, it was found that the mutation rate of the region that does not bind calcium is almost twice as high as the mutation rate of the three homologous regions that do bind calcium.     

Structural similarities between the Ca2+-dependent regulatory proteins of 3':5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase.

Results of studies of the Ca2+-dependent protein modulator of 3':5'-cyclic nucleotide phosphodiesterase isolated from bovine brain are presented which show its structural similarity to the Ca2+-binding subunit of muscle troponin. Both proteins have blocked NH2 termini, similar and characteristic ultraviolet absorption spectra, similar Ca2+-binding properties, very similar amino acid compositions, and co-migrate on sodium dodecyl sulfate-polyacrylamide gels. The primary structures of selected tryptic peptides isolated from bovine brain modulator protein are similar or identical with regions of the primary sequences of rabbit skeletal muscle and bovine cardiac muscle troponin C. Bovine brain modulator protein contains and unidentified ninhydrin-positive basic compound not found in muscle troponin C. An improved procedure is presented which yields 40 to 70 mg of modulator protein per kg of bovine brain.     

The amino-acid sequence of troponin C from frog skeletal muscle.

The primary structure of the troponin C from skeletal muscle of the frog Rana esculenta has been determined. The amino acid sequence was deduced from amino acid determinations of peptides obtained after cleavage with cyanogen bromide. Overlapping peptides were isolated from tryptic digests of performic-acid-oxidized troponin C and phthalylated performic-acid-oxidized troponin C. All overlaps have been determined except for the Arg-Ile sequence at position 103--104, which has been obtained by comparison with homologous troponins C. Frog troponin C consists of one polypeptide chain containing 152 amino acids. The calculated molecular weight is 18299. There is a single cysteine residue at position 101 and a single tyrosine residue at position 112. No histidine or tryptophan residues are present. The amino-terminal amino acid is N-acetylated. The homology of frog troponin C with other skeletal and cardiac troponin C is briefly discussed.     

Primary structure of rabbit skeletal muscle glycogen synthase deduced from cDNA clones

The complete amino acid sequence of rabbit skeletal muscle glycogen synthase was deduced from cDNA clones with a composite length of 3317 bp. An mRNA of 3.6 kb was identified by Northern blot analysis of rabbit skeletal muscle RNA. The mRNA coded for a protein of 734 residues with a molecular weight of 83,480. The deduced NH2-terminal and COOH-terminal sequences corresponded to those reported for the purified protein, indicating the absence of any proteolytic processing. At the nucleotide level, the 5' untranslated and coding regions were 79 and 90% identical for rabbit and human muscle glycogen synthases, whereas the 3' untranslated regions were significantly less similar. The enzymes had 97% amino acid sequence identity. Interestingly, the NH2 and COOH termini of rabbit and human muscle glycogen synthase, the regions of phosphorylation, showed the greatest sequence variation (15 of 19 mismatches and two insertion/deletion events), which may indicate different evolutionary constraints in the regulatory and catalytic regions of the molecule.     

The amino acid sequence of rabbit cardiac troponin I.

The complete amino acid sequence of troponin I from rabbit cardiac muscle was determined by the isolation of four unique CNBr fragments, together with overlapping tryptic peptides containing radioactive methionine residues. Overlap data for residues 35-36, 93-94 and 140-145 are incomplete, the sequence at these positions being based on homology with the sequence of the fast-skeletal-muscle protein. Cardiac troponin I is a single polypeptide chain of 206 residues with mol.wt. 23550 and an extinction coefficient, E 1%,1cm/280, of 4.37. The protein has a net positive charge of 14 and is thus somewhat more basic than troponin I from fast-skeletal muscle. Comparison of the sequences of troponin I from cardiac and fast skeletal muscle show that the cardiac protein has 26 extra residues at the N-terminus which account for the larger size of the protein. In the remainder of sequence there is a considerable degree of homology, this being greater in the C-terminal two-thirds of the molecule. The region in the cardiac protein corresponding to the peptide with inhibitory activity from the fast-skeletal-muscle protein is very similar and it seems unlikely that this is the cause of the difference in inhibitory activity between the two proteins. The region responsible for binding troponin C, however, possesses a lower degree of homology. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50072 (20 pages), at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1976) 153, 5.     

Human skeletal muscle proteins

Summary The primary structure of the major component of human skeletal muscle troponin C has been established. The troponin C was purified by ammonium sulphate and isoelectric fractionation, followed by two chromatographic steps on DEAE Sephadex. The sequence was determined from the different overlapping enzymic peptides and by dansyl-Edman degradation. The only difference between rabbit skeletal muscle troponin C and the major component of human skeletal troponin C was found at position 112: Ala (rabbit), Pro (human). The partial amino acid sequence of the first 86 residues of the minor component of human skeletal troponin C was found to resemble the troponin C from bovine cardiac muscle. The only difference between them, has tentatively been located at position 62: Glu (human), Asp (bovine). These similarities suggest that troponin C is, from the point of view of molecular evolution, one of the most conservative proteins so far studied.     

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.