Abstract
Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.
Highlights
The mollusc shell consists of two calcium carbonate polymorphs; the outer layer of calcite and the inner iridescent aragonite, or nacre [1]
Perlucin Splice Variants Based on the amino acid sequence of purified Perlucin degenerate primers were designed and used in reverse transcriptase PCR (RT-PCR) with cDNA derived from mRNA of the mantle epithelia of H. laevigata
The mass spectrometry data of purified native Perlucin digested with trypsin clearly showed that these variants are present in the shell of H. laevigata, since a NH2-DSLHANLQQMDSLHANLQQR-COOH peptide was identified in protein spots with apparent molecular weights of 25 kDa and 20 kDa, but not in the spots 5 and 6 appearing at around 15 kDa (Figure 3)
Summary
The mollusc shell consists of two calcium carbonate polymorphs; the outer layer of calcite and the inner iridescent aragonite, or nacre [1]. In early shell formation there is a predominately irregular growth of calcite with low expression levels of proteins, followed by more regular growth of calcite on top of the first nacre layer controlled by the organic matrix and mediated by a significant increase of protein expression [11] Far those water-soluble proteins of abalone nacre that have been studied include Perlucin [12,13,14], Perlustrin [13,15], Perlwapin [16], Perlinhibin [17], Perlbikunin from H. laevigata and Lustrin A [18,19,20,21] AP7, AP8 [22], AP24 [20,23,24,25] from H. rufescens. The physiological function of this activity has not yet been investigated
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