We have cloned and sequenced a novel Bombyx mori gene that encodes a protein having a high degree of homology with other known troponin C (TnC) proteins. The amino acid sequence, DX[DN]X[DSG]X6E, a highly conserved putative Ca2+-binding motif found in loops within the globular domains of previously identified TnC proteins, is also present in BmTnC. We have expressed and purified to homogeneity a His-tagged BmTnC fusion protein having a molecular weight of approximately 21.6 kDa. We have used this purified fusion protein to produce polyclonal antibodies against BmTnC for Western blot analyses. These analyses have revealed that BmTnC is expressed in the larval head, the Malpighian tubule, the epidermis, the testis, and the gut, as has been confirmed by immunohistochemistry. In addition, real-time reverse transcription/polymerase chain reaction has shown that BmTnC mRNA levels differ substantially among these tissues. Our findings indicate that BmTnC is selectively expressed in the muscular tissues of the silkworm, including portions of the head, the Malpighian tubule, the body wall, and the gut.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Adamcova M, Sterba M, Simunek T, Potacova A, Popelova O, Gersl V (2006) Myocardial regulatory proteins and heart failure. Eur J Heart Fail 8:333–342
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Babu A, Su H, Ryu Y, Gulati J (1992) Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination, by genetic engineering. J Biol Chem 267:15469–15474
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Fyrberg C, Parker H, Hutchison B, Fyrberg E (1994) Drosophila melanogaster genes encoding three troponin-C isoforms and a calmodulin-related protein. Biochem Genet 32:119–135
Garcia Gonzalez MJ, Dominguez Rodriguez A (2006) Pharmacologic treatment of heart failure due to ventricular dysfunction by myocardial stunning: potential role of levosimendan. Am J Cardiovasc Drugs 6:69–75
Gordon AM, Homsher E, Regnier M (2000) Regulation of contraction in striated muscle. Physiol Rev 80:853–924
Grabarek Z, Leavis PC, Gergely J (1986) Calcium binding to the low affinity sites in troponin C induces conformational changes in the high affinity domain. A possible route of information transfer in activation of muscle contraction. J Biol Chem 261:608–613
Gross M, Kumar R (1990) Physiology and biochemistry of vitamin dependent calcium binding proteins. Am J Physiol 259:F195–F209
Gusev NB (1986) Troponin from the myocardium and skeletal muscles: structure and properties. Biokhimiia 51:1993–2009
Herranz R, Diaz-Castillo C, Nguyen TP, Lovato TL, Cripps RM, Marco R (2004) Expression patterns of the whole troponin C gene repertoire during Drosophila development. Gene Expr Patterns 4:183–190
Herzberg O, James MN (1985) Structure of the calcium regulatory muscle protein troponin-C at 2.8 Å resolution. Nature 313:653–659
Herzberg O, Moult J, James MN (1986) A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction. J Biol Chem 261:2638–2644
Joseph S, David WR (2002) Expression of cloned genes in E.coli using IPTG-inducible promoters. In: Joseph S, David WR (eds) Molecular cloning: a laboratory manual. Expression of cloned genes in Escherichia coli, vol 3, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 15.14–15.19
Kawai M, Lee JA, Orchard CH (2000) Effects of the Ca2+ sensitizer EMD 57033 on intracellular Ca2+ in rat ventricular myocytes: relevance to arrhythmogenesis during positive inotropy. Clin Sci (Lond) 99:547–554
Kawasaki H, Kretsinger RH (1995) Calcium-binding proteins 1: EF-hands. Protein Prof 2:305–490
Kumar S, Tamura K, Nei, M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680-685
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C[T]) method. Methods 25:402–408
Ming MJ, Hu D, Chen HS, Liu LM, Nan X, Hua CH, Lu RQ (2000) Effect of MCI-154, a calcium sensitizer, on calcium sensitivity of myocardial fibers in endotoxic shock rats. Shock 14:652–656
Moncrief ND, Kretsinger RH, Goodman M (1990) Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J Mol Evol 30:522–562
Nakayama S, Moncrief ND, Kretsinger RH (1992) Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories. J Mol Evol 34:416–448
Qiu F, Lakey A, Agianian B, Hutchings A, Butcher GW, Labeit S, Leonard K, Bullard B (2003) Troponin C in different insect muscle types: identification of two isoforms in Lethocerus, Drosophila and Anopheles that are specific to asynchronous flight muscle in the adult insect. Biochem J 371:811–821
Schreier T, Kedes L, Gahlmann R (1990) Cloning, structural analysis, and expression of the human slow twitch skeletal muscle/cardiac troponin C gene. J Biol Chem 265:21247–21253
Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385
Sundaralingam M, Bergstrom R, Strasburg G, Rao ST, Roychowdhury P, Greaser M, Wang BC (1985) Molecular structure of troponin C from chicken skeletal muscle at 3-angstrom resolution. Science 227:945–948
Wang JM, Wilson PB, Kumar S, Pye D, Hunter RD (1994) Quantitation of endothelial cell specific protein E-9 employing a single monoclonal antibody in an indirect sandwich ELISA. J Immunol Methods 171:55–64
Wang X, Li MX, Sykes BD (2002) Structure of the regulatory N-domain of human cardiac troponin C in complex with human cardiac troponin I147–163 and bepridil. J Biol Chem 277:31124–31133
Wilkins MR, Lindskog I, Gasteiger E, Bairoch A, Sanchez JC, Hochstrasser DF, Appel RD (1997) Detailed peptide characterization using PEPTIDEMASS—a world-wide-web-accessible tool. Electrophoresis 18:403–408
This work was supported by grants from the National High Technology Research and Development Program of China (no. 2005AA206120) and the National Basic Research Program of China (no. 2005CB121006).
About this article
Cite this article
Chen, J., Chen, J., Gai, Q. et al. Molecular characterization and immunohistochemical localization of a novel troponin C during silkworm development. Cell Tissue Res 331, 725 (2008). https://doi.org/10.1007/s00441-007-0516-1
- Troponin C
- Expression pattern
- Bombyx mori (Insecta)