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Peptides
Volume 28, Issue 3, March 2007, Pages 566-573
 
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doi:10.1016/j.peptides.2006.12.005    
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Copyright © 2006 Elsevier Inc. All rights reserved.

Significance of the N- and C-terminal regions of CAP-1, a cuticle calcification-associated peptide from the exoskeleton of the crayfish, for calcification

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Hirotaka Inouea, Tsuyoshi Ohiraa and Hiromichi NagasawaCorresponding Author Contact Information, a, E-mail The Corresponding Author

aDepartment of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan


Received 6 October 2006; 
revised 30 November 2006; 
accepted 1 December 2006. 
Available online 15 December 2006.

Abstract

Calcification-associated peptide (CAP)-1 is considered to play an important role in calcification of the exoskeleton of the crayfish, Procambarus clarkii. In this study, in order to clarify the molecular mechanism of calcification, we constructed expression systems of recombinant molecules of CAP-1 and its related peptides in Escherichia coli, and verified the structure–activity relationship of these peptides. The inhibitory assay on calcium carbonate precipitation and the calcium-binding assay showed that the C-terminal acidic region was most important for both activities. The CD spectra of these peptides varied depending on calcium concentration and showed that the N-terminal region is associated with the peptide conformation. These results indicate that the C-terminal part of CAP-1 may concentrate calcium ions for nucleation and/or interact with calcium carbonate precipitate to control the growth and that the N-terminal part contribute to maintaining the peptide conformation.

Keywords: Biomineralization; Calcification; Calcium binding; Cuticle peptide; Exoskeleton

Article Outline

1. Introduction
2. Material and methods
2.1. Construction of expression plasmids
2.2. Expression and purification of recombinant peptides
2.3. Amino acid sequence and mass spectral analyses
2.4. Assay for inhibitory activity on calcium carbonate precipitation
2.5. Calcium binding assay
2.6. CD spectral analysis
3. Results
3.1. Expression and purification of recombinant CAP-1 and the related peptides
3.2. Inhibitory activity on calcium carbonate precipitation
3.3. Calcium-binding ability
3.4. CD spectral analysis
4. Discussion
Acknowledgements
References







Corresponding Author Contact InformationCorresponding author. Tel.: +81 3 5841 5132; fax: +81 3 5841 8022.

Peptides
Volume 28, Issue 3, March 2007, Pages 566-573
 
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