Abstract
BackgroundThe shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date.MethodsThe shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing.ResultsWe identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins.ConclusionsThe H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.
Highlights
The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years
We cleaned most H. laevigata shells with sodium hypochlorite prior demineralization to destroy and remove contaminating organic material adhering to the shell surface
We took great care in cleaning the surface of the shells before extraction of the matrix, indicating that these proteins were an integral part of the shell structure and are difficult or impossible to remove without causing damage. This was shown to be the case with the shell of the brachiopod Magellania venosa [62], where we succeeded in significantly reducing the level of intracellular proteins when we treated powdered shell particles for 24 h with hypochlorite, and lost some interesting proteins with some features characteristic of shell proteins, probably by removal of a large part of the extra-crystalline matrix
Summary
The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. The inner mother-of-pearl layer, or nacre, is characterized by thin intercalated plates and has attracted much more interest as a model in biomaterials and biomineralization research than the prismatic layer. This is due to its extraordinary toughness and fracture resistance conferred by the arrangement of individual aragonite crystals which are connected by mineral bridges and enclosed by a thin layer of organic matrix [3,4,5,6]. Isolated H. rufescens organic shell matrix was shown to control nucleation, crystal orientation, the nature of the calcium carbonate polymorph deposited [8,9,10,11], and to act as an adhesive between the aragonitic plates [12]
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