Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Molecular characterization and immunohistochemical localization of a novel troponin C during silkworm development

  • 123 Accesses

  • 7 Citations


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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11


  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Gordon AM, Homsher E, Regnier M (2000) Regulation of contraction in striated muscle. Physiol Rev 80:853–924

  8. 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

  9. Gross M, Kumar R (1990) Physiology and biochemistry of vitamin dependent calcium binding proteins. Am J Physiol 259:F195–F209

  10. Gusev NB (1986) Troponin from the myocardium and skeletal muscles: structure and properties. Biokhimiia 51:1993–2009

  11. 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

  12. Herzberg O, James MN (1985) Structure of the calcium regulatory muscle protein troponin-C at 2.8 Å resolution. Nature 313:653–659

  13. 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

  14. 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

  15. 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

  16. Kawasaki H, Kretsinger RH (1995) Calcium-binding proteins 1: EF-hands. Protein Prof 2:305–490

  17. Kumar S, Tamura K, Nei, M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680-685

  19. 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

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385

  26. 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

  27. 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

  28. 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

  29. 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

Download references

Author information

Correspondence to Yaozhou Zhang.

Additional information

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).

Rights and permissions

Reprints and Permissions

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

Download citation


  • Troponin C
  • Development
  • Expression pattern
  • Immunohistochemistry
  • Bombyx mori (Insecta)