Primary structure and functional expression of h-caldesmon complementary DNA

Recently, the two Mr forms of caldesmon (Mr's in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified. h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells. In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence. This sequence predicts a protein of 771 amino acids with a Mr of 88,743. The central portion of this sequence is composed of a 10-fold repeat of conserved amino acid sequence containing 13-15 amino acids. Further, a recombinant protein produced in Escherichia coli containing the full-length h-caldesmon cDNA has been characterized. Although the Mr of h-caldesmon predicted from amino acid sequence is 88,743, native and recombinant proteins show the same mol. wt. with 150kDa as measured by SDS-PAGE. This discrepancy may be due to the acidic amino acid-rich sequences at the N-terminal and central portions. A recombinant protein produced in E. coli possesses calmodulin-, F-actin- and tropomyosin-binding abilities in common with the native h-caldesmon.     

Caldesmon structure and function: sequence analysis of a 35 kilodalton actin- and calmodulin-binding fragment from the C-terminus of the turkey gizzard protein

We have determined the amino acid sequence of a 35 kDa proteolytic fragment ("CaD35") derived from the C-terminus of turkey gizzard caldesmon. This 239-residue peptide contains binding sites for actin and calmodulin. Residues 1-96 of CaD35 comprise "CaD15", an actin-binding subfragment which we previously showed to resemble the tropomyosin-binding segment of troponin T. The remainder of the CaD35 sequence shows no significant similarity to other proteins. Residues 111-128 may form a basic, amphipathic helix which interacts with calmodulin.     

Amino acid sequence of a 15 kilodalton actin-binding fragment of turkey gizzard caldesmon: similarity with dystrophin, tropomyosin and the tropomyosin-binding region of troponin T

We have determined the amino acid sequence of a 15 kDa actin-binding fragment of turkey gizzard caldesmon. The 96-residue fragment contains 29 acidic and 29 basic residues, and is predicted to have an extended helical conformation stabilized by numerous internal salt bridges. CaD15 bears some resemblance to dystrophin, tropomyosin and several other proteins, but is most strikingly similar to the tropomyosin-binding segment of troponin T.     

Complete amino acid sequence of subunit e of rat liver mitochondrial H(+)-ATP synthase.

Subunit e of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high-performance liquid chromatography. The amino acid sequence of the subunit was determined by automated Edman degradation of the whole protein and derived peptides. The nucleotide sequence of the import precursor of subunit e of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 289 nucleotides including a coding region for the import precursor of subunit e and noncoding regions on the 5'- and 3'-sides. The possible import precursor of subunit e and its mature polypeptide deduced from the open reading frame consisted of 71 and 70 amino acid residues with molecular weights of 8254 and 8123, respectively. Subunit e is a basic hydrophilic protein with an isoelectric point of 9.78. The sequence of the rat subunit e is highly homologous with that of subunit e of bovine heart, but has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase. The function of subunit e is unknown. However, a homology search in the database of the National Biomedical Research Foundation revealed that residues 34-65 of subunit e are homologous with residues 90-117 of troponin T, and with residues 529-561 of h-caldesmon and residues 289-319 of l-caldesmon, which are the homologous sequences corresponding to the Ca(2+)-dependent tropomyosin-binding region of troponin T.     

Interactions between nebulin-like motifs and thin filament regulatory proteins

Nebulin (600-900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of approximately 35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.     

Structure–function relationships of molluscan troponin T revealed by limited proteolysis

Molluscan troponin regulates muscle contraction through a novel Ca²⁺-dependent activating mechanism associated with Ca²⁺-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin I. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin I, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca²⁺-dependent activation of the Mg-ATPase activity of rabbit-actomyosin–scallop-tropomyosin. In addition, the actin–tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca²⁺ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin–tropomyosin filaments and is essential for regulation.     

Molecular cloning of human cardiac troponin I using polymerase chain reaction

We have used the polymerase chain reaction (PCR) to synthesise a cDNA encoding part of human cardiac troponin I. Amplification was achieved using fully degenerate sets of oligonucleotides corresponding to conserved regions of amino acid sequence identified in other troponin I isoforms. The cloned PCR fragment was subsequently used to isolate full-length cDNAs from a cardiac cDNA library. We describe the approach, as a general cloning strategy starting from limited amino-acid sequence data and report the cloning, and complete amino acid sequence of human cardiac troponin I. Analysis of human development using these clones demonstrates early expression of this gene in the heart.     

The expression of multiple forms of troponin T in chicken-fast-skeletal muscle may result from differential splicing of a single gene

Troponin T isolated from chicken fast skeletal muscle has been shown to be present in three different molecular forms, one in breast and two in leg muscle. The three forms differ in both size and charge. Troponin T from breast muscle has a molecular mass of 33.5 kDa and a pI of about 7. Of the two leg muscle forms the larger has a molecular mass of 30.5 kDa and a pI of about 8.5 and the smaller a molecular mass of 29.8 kDa and a pI of about 10. Considerably more heterogeneity has been found in the leg than in the breast muscle proteins although this is not reflected in their N-terminal sequences. The reason for this is not clear. Troponin T from breast or leg muscle can be phosphorylated with troponin T kinase at the single serine residue at the N-terminus. No difference in the rate or extent of phosphorylation could be found between proteins from breast or leg muscle. The three proteins have been shown to differ only in the amino acid sequence of their N-terminal tryptic peptides. These peptides are of different length, that from breast troponin T being 58 residues and those from leg troponin T being 36 and 42 residues, these differences account for the difference in molecular mass of the parent proteins. Despite this difference the sequence of the first 12 and last 14 residues is identical in all three N-terminal peptides. The remainder of the sequence of the smallest peptide is also repeated in the other two but they each contain an extra piece of unique sequence. On the basis of these sequences it is proposed that chicken troponin T is coded for by a single gene containing, at the 5' end, a number of small exons and that three different mRNA molecules may be produced by alternative pathways of RNA splicing. The possible significance of these N-terminal sequence variations is discussed.     

转录因子DREB1A基因的克隆与植物表达载体的构建

根据GenBank中已发表的转录因子DREB1A基因的cDNA序列设计并合成了一对引物,通过RT-PCR的方法从低温处理的拟南芥总RNA中扩增出DREB1A基因的全长cDNA片段。将其克隆到pMD18 T-vector中。经测序证明该片段与GenBank上报道的序列具有99.8%的同源性。2个碱基的置换导致了一处氨基酸的差异,但这一氨基酸并不在基因的功能结构域上,推测其不会影响基因功能。以植物表达载体pBch为基础,构建了由组成型启动子35S调控的DREB1A基因的植物表达载体pBDR35S,为利用DREB1A基因改良植物抗逆性奠定了物质基础。     

Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis.

A cDNA expression library prepared from mRNA of Haemaphysalis longicornis (H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128-148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue.     

Primary structure of human plasma fibronectin--characterization of the 6,000 dalton C-terminal fragment containing the interchain disulfide bridges

The carboxy terminal fragment of human plasma fibronectin has been isolated after tryptic digestion and separation by DEAE-cellulose chromatography and gel filtration on Sephadex G-50. It has a molecular weight of 6,000 which changes to 3,000 after reduction indicating that the fragment is a dimer. We have determined the amino acid sequence of the 6kDa fragment and showed that it contains 26 residues including two half-cystines which form two interchain disulfide bridges. The 6kDa fragment is not phosphorylated as in bovine fibronectin although its amino acid sequence is identical to that reported for bovine plasma fibronectin. When compared to the sequence deduced from a rat cDNA, one amino acid substitution can be found. It appears that the carboxyl end of fibronectin is highly conserved among species.     

Isolation and Characterization of Goldfish cdc2, a Catalytic Component of Maturation-Promoting Factor

We have isolated a cdc2 cDNA from a library constructed from immature goldfish oocytes. The isolated clone has a PSTAVR sequence, instead of the PSTAIR sequence common to cdc2 in other species. Its product was characterized by monoclonal antibodies against its C-terminal amino acid sequence. The antibodies recognized an anti-PSTAIR-reactive 35 kDa protein in immature oocyte extracts, which was not recognized by anti-goldfish cdk2 antibody. In addition to the 35 kDa cdc2, mature oocytes contained a 34 kDa cdc2, which was a component of MPF purified from carp eggs. Upon gel filtration column, the 35 kDa cdc2 migrated at monomeric position, while the 34 kDa cdc2 migrated at around 100 kDa, where cyclin B also comigrated. These results strongly suggest that the 35 kDa protein is monomeric inactive cdc2, while the 34 kDa protein is cyclin B-bound active cdc2. The finding that the 35 kDa inactive cdc2 does not form a complex with any other proteins in immature oocytes is in contrast to the situation in Xenopus and starfish, in which cdc2-cyclin B complex exists already as pre-MPF in immature oocytes.     

Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.     

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.     

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).     

Cloning and expression of a smooth muscle caldesmon

Caldesmon is a smooth muscle and nonmuscle regulatory protein that interacts with actin, myosin, tropomyosin, and calmodulin. Two overlapping clones, isolated from a chicken oviduct cDNA plasmid library and a chicken gizzard cDNA lambda NM1149 library, were used to generate a 4108-base pair sequence coding for one caldesmon. Expression of the coding sequence confirms this is one of the large smooth muscle caldesmons. The deduced protein molecular weight is 86.974, significantly less than the molecular weights estimated by sodium dodecyl sulfate gel electrophoresis. The protein has a high content of Gly, Lys, Arg, and Ala; there are two cysteine residues, one at either end of the molecule. Comparison with the Protein Identification Resource database demonstrates a similarity with a tropomyosin binding domain of troponin T, but none with any calmodulin or actin binding proteins. The center of the protein has an 8-fold repeat of a 13 amino acid sequence whose general motif is -Glu3-(Lys/Arg)2-Ala2-Glu2-(Lys/Arg)1-X-(Lys/Arg)1-Ala1-, where X is Glu, Gln, or Ala. Comparison with peptide sequences from a chymotryptic fragment that binds actin and calmodulin places this domain on the C terminus of caldesmon adjacent to the troponin T similarity. A tentative map of the major binding domains is proposed on the basis of available data.     

Characterization of two antifungal endochitinases from barley grain

A basic chitinase (chitinase T, EC 3.2.1.14, molecular mass 33 kDa, pI 9.8) was isolated and compared with a previously described chitinase (chitinase C, molecular mass 28 kDa, pI 9.7). The two chitinases were isolated in homogeneous form from barley ( Hordeum vulgare L.) Bomi mutant 1508 grains either by two cation exchange steps or by one affinity step followed by cation exchange. Both chitinases are endochitinases with specific activities of 168 and 54 nkat (mg protein)⁻¹ for chitinase T and chitinase C, respectively. Both inhibit the growth of Trichoderma viride efficiently. The lysozyme activity of both chitinases is 10⁴ times lower than that of hen egg-white lysozyme as measured by lysis of cell walls of Micrococcus lysodeikticus . The amino acid composition and two partial amino acid sequences of chitinase T were determined. A 23 residue sequence of the N-terminal domain of chitinase T, which was not present in chitinase C, showed 73% identity with domain B of wheat germ lectin and 65% identity with the N-terminal domain of an endochitinase from bean leaves (deduced from cDNA). A 9 amino acid sequence of a cyanogen bromide fragment of chitinase T was identical with a cDNA deduced sequence of a barley aleurone endochitinase but differed in one residue from chitinase C. Generally, the two grain chitinases have physico-chemical and enzymatic properties similar to the plant leaf chitinases characterized. Both chitinases are localized in the aleurone layer and starchy endosperm of developing and germinating grain, but not in the embryo. The appearance of chitinases T and C at a late state of grain development suggests a role for these enzymes as a defense against fungi in the quiescent and germinating grain.     

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.