Marine Biotechnology

, Volume 13, Issue 5, pp 955–962

Proteomic Identification of Novel Proteins from the Calcifying Shell Matrix of the Manila Clam Venerupis Philippinarum

  • Benjamin Marie
  • Nolwenn Trinkler
  • Isabelle Zanella-Cleon
  • Nathalie Guichard
  • Michel Becchi
  • Christine Paillard
  • Frédéric Marin
Original Article

DOI: 10.1007/s10126-010-9357-0

Cite this article as:
Marie, B., Trinkler, N., Zanella-Cleon, I. et al. Mar Biotechnol (2011) 13: 955. doi:10.1007/s10126-010-9357-0
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Abstract

The shell of the Manila clam Venerupis philippinarum is composed of more than 99% calcium carbonate and of a small amount of organic matrix (around 0.2%). In this study, we developed one of the first proteomic approaches applied to mollusc shell in order to characterise the matrix proteins that are believed to be essential for the formation of the biomineral. The insoluble organic matrix, purified after demineralisation of the shell powder with cold acetic acid (5%), was digested with trypsin enzyme and then separated on nano-LC prior to nanospray/quadrupole time-of-flight analysis. MS/MS spectra were searched against the above 11,000 EST sequences available on the NCBI public database for Venerupis. Using this approach, we were able to identify partial or full-length sequence transcripts that encode for shell matrix proteins. These include three novel shell proteins whose sequences do not present any homologous proteins or already described domains, two putative protease inhibitor proteins containing Kazal-type domains, and a putative Ca2+-binding protein containing two EF-hand domains. Biomineral formation and evolutionary implications are discussed.

Keywords

BiomineralizationCalcium carbonateProteomicsMollusc shellOrganic matrixCalcium-binding proteinSerine protease inhibitor

Abbreviations

AA

Amino acid

AIM

Acid-insoluble matrix

ISMP

Insoluble matrix shell protein

SEM

Scanning electron microscopy

ESTs

Expressed sequence tags

Supplementary material

10126_2010_9357_MOESM1_ESM.doc (26 kb)
Additional Data 1An alignment of AM851246, AM877557 and the deduced putative clustered sequence (Put_clust) of IMSP-1. Predicted signal peptide is underlined. Peptides identified by MS/MS are in red. GenBank numbers are indicated on the left. Asterisk indicates the stop codon. AA positions that are variable in the different sequences are in blue (DOC 25 kb)
10126_2010_9357_MOESM2_ESM.doc (39 kb)
Additional Data 2Alignment of the deduced putative clustered sequence (Put_clust) of IMSP-1 and other mollusc and cnidarian putative proteins presenting high sequence similarities. Predicted signal peptides are underlined. Asterisks indicate the stop codon. The positions shaded in black indicate where more than 80% of the residues are identical and in grey where at least 60% of the residues are identical or present biochemical similarities. GenBank numbers and species name are indicated on the left (DOC 39 kb)
10126_2010_9357_MOESM3_ESM.doc (26 kb)
Additional Data 3Sequence analysis of ISMP-2. a The protein sequence is deduced from the translated sequences of the entry AM876908. The predicted signal peptide is underlined. Peptides identified by MS/MS are in red. Asterisk indicates the stop codon. b Alignment of ISMP-2 with two other putative proteins from V. phillipinarum presenting high sequence similarities. Predicted signal peptides are underlined. The positions shaded in black indicate where AA are identical and in grey where at least two AA are identical or present biochemical similarities. GenBank numbers are indicated on the left (DOC 26 kb)
10126_2010_9357_MOESM4_ESM.doc (31 kb)
Additional Data 4Kazal-type-containing protein IMSP-4 and IMSP-6. a Alignment of AM874135, AM875391, AM875417 and the deduced putative clustered sequence (Put_clust) of IMSP-4. Peptide identified by MS/MS is in red. GenBank numbers are indicated on the left. Asterisk indicates the stop codon. AA positions that are variable with the different sequences are in blue. The Kazal-type domain is shaded in grey. b Alignment of ISMP-6 (AM876906) and other mollusc putative proteins presenting high sequence similarities, signal peptides and two Kazal-type domains. Predicted signal peptides are underlined. The positions shaded in black indicate where more than 80% of the residues are identical and in grey where at least 60% of the residues are identical or present biochemical similarities. GenBank and Swiss-Prot numbers are indicated on the left (DOC 31 kb)

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Benjamin Marie
    • 1
    • 2
  • Nolwenn Trinkler
    • 3
  • Isabelle Zanella-Cleon
    • 4
  • Nathalie Guichard
    • 1
    • 2
  • Michel Becchi
    • 4
  • Christine Paillard
    • 3
  • Frédéric Marin
    • 1
    • 2
  1. 1.UMR 5561 CNRS BiogéosciencesUniversité de BourgogneDijonFrance
  2. 2.Université de BourgogneDijonFrance
  3. 3.UMR 6539 UBO/IUEM, LEMARPlouzanéFrance
  4. 4.IFR 128 BioSciences Gerland-Lyon Sud, UMR 5086 CNRS, IBCPUniversité de Lyon 1LyonFrance