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Calcification in the Shell of the Freshwater Bivalve Unio pictorum

Benjamin Marie1*, Nathalie Guichard1, Jean-Paul Pais De Barros2, Gilles Luquet1 and Frédéric Marin1*

1UMR CNRS 5561 Biogéosciences, University of Burgundy, 6 Bd. Gabriel, F-21000 Dijon, France. 2U 498 INSERM, University of Burgundy,7 Bd. Jeanne d’Arc F-21079 Dijon, France.

benjamin.marie@u-bourgogne.fr*, frederic.marin@u-bourgogne.fr*

ABSTRACT

The scope of the present study is to elucidate the primary structure of the shell proteins of the mollusk Unio pictorum. This freshwater bivalve exhibits an aragonitic shell with nacro-prismatic microstructures. The acetic acid-soluble matrix of the nacreous layer has been extracted, and analyzed on standard polyacrylamide gel under denaturing conditions. Five main proteins can be observed. Their respective apparent molecular weight is 95, 50, 29, 14 and 10 kDa. The three high molecular weight bands are acidic, glycosylated and bind calcium in vitro. These proteins are purified by preparative SDS-PAGE. In addition, these fractions are tested to check their ability to modify the shape of CaCO3 crystals. The biochemical properties of these proteins can be compared to that of other proteins of nacreous mollusks, such as Pinctada sp., Pinna nobilis, Haliotis sp. and Turbo marmoratus.

INTRODUCTION

Among metazoans, the shells secreted by mollusks are a remarkable example of a natural composite biomaterial, synthesized at ambient temperature. Despite numerous detailed studies carried out on molluscan shell biomineralization, it still remains obscure how mollusks produce and shape their shells. Various taxa of mollusks exhibit a shell with a nacreous layer characterized by highly oriented calcium carbonate crystals of aragonite embedded in a small amount of organic material (1-5% weight). At macroscale, this matrix is, among others, responsible for the remarkable fracture toughness of the shell (Weiner, 1986). At molecular scale, it is mainly constituted of proteins, polysaccharides and lipids. It has been shown to regulate the crystal nucleation, the crystal size and morphology, and to act as a growth inhibitor (Weiner and Addadi, 1997).

Recently, a number of shell matrix proteins that are involved in nacre formation have been characterized among gastropods and bivalves (Marin and Luquet, 2004). In this latter group, all the molecular data published so far concern the pteriomorphids. To date, very little is known on the shell macromolecular

Biomineralization: from Paleontology to Materials ScienceProceedings of the 9th International Symposium on Biomineralization,

(Eds.) Arias, J.L., Fernández, M.S.Editorial Universitaria, Santiago, Chile, 2007, pp. 273-280.

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constituents of Paleoheterondont bivalves. Our knowledge is mostly limited to the extrapallial fluid components (Moura et al., 2000; Lopes-Lima et al., 2005). The shell of the Unionidae is indeed entirely aragonitic, and made of two mineralized layers; the external one is made of prisms, and the internal layer is nacreous (Checa et al., 2000).

The aim of the present study is to investigate the protein content of nacre from the freshwater painter’s mussel Unio pictorum.

MATERIALS AND METHODS

Shell matrix extraction Lake mussels with 10-16 mm of shell length (Fig. 1-1), belonging to the species Unio pictorum, were collected from the canal of Burgundy (Dijon, France). The shells were immersed in 1% (v/v) NaClO to remove the periostracum and superficial contaminants. The shells were mechanically abraded, cleaned and crushed. The powder of the two aragonitic layers (nacreous and prismatic) or of the nacreous layer alone was decalcified overnight at 4°C in cold dilute acetic acid (10% v/v). The entire extract was directly centrifuged at 3900 g (15 min, 4°C). The supernatant solution was filtered (5 μm) before being concentrated by ultra-filtration (cut-off 10 kDa). The concentrated solution (about 5 mL) was dialyzed against MilliQ water (3 days) before being freeze-dried and weighed.

Figure 1. Shell of Unio pictorum.1) General view. 2) SEM internal view of the nacre (scale bar 30 µm). 3) Transversal view (scale bar 20 μm). 4) 15% SDS-PAGE profile of ASM stained by CBB. A: molecular weight markers (116, 97.4, 65, 45, 31, 21, 14.4 et 6.5 kDa) ; B : ASM (95, 50, 29, 14 and

10 kDa).

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Mono and two-dimensional PAGE

The macromolecules of the Acido-Soluble Matrix (ASM) were fractionated by denaturing SDS-PAGE (Mini Protean III, Bio-Rad). Gels were subsequently stained by Coomassie brillant blue (CBB), silver nitrate, alcian blue, PAS, ‘Stains-All’ or blotted on a polyvinylidene difluoride membrane for Ca45-binding test or for Western blot analysis, as described by Marin et al. (2005). The ASM was also fractionated on 2-D electrophoresis. The first dimension was performed by isoelectric focusing in IPG strips (pH 3-10) in presence of ampholytes. The second dimension was subsequently run under denaturing conditions (4-10% SDS-PAGE).

Protein purification

The denatured matrix was fractionated on a preparative SDS-PAGE as described by Marin et al. (2001). Fractions containing purified proteins were detected immunologically by dot-blot performed with the anti-nacre of P. nobilis matrix antiserum. The purity of the fractions was assessed on a mini-SDS polyacrylamide gel, which was subsequently stained with silver nitrate. Fractions of the same proteins were pooled, thoroughly dialyzed against MilliQ water and freeze-dried.

Protein-calcite interaction test

Synthetic calcite crystals were grown by slow diffusion of ammonium bicarbonate in 500 μl of 10 mM calcium chloride solution (Albeck et al., 1993) in presence of different concentrations of ASM (24 h, 4°C). Blank tests were performed without any macromolecule. Dried crystals were observed by scanning electron microscopy (JEOL 6400).

RESULTS

Unio pictorum shell consists of an outer aragonitic prismatic layer and an inner nacreous layer with the prismatic layer comprising only 10-30% of the total thickness. Prisms are fibrous polycrystalline aggregates. Transition to nacre is gradual. In Unionidae, ‘the first nacreous tablets grow by epitaxy onto the distal ends of the prism fibres’ (Checa, 2000) The inner surface of the nacreous layer shows a terraced disposition of aragonite sheets, which grow towards the shell margin. The shape of aragonite tablets is rhombic (Fig. 1-2 and 1-3).

The ASM represents 0.04 % of the dry weight of the shells of Unio pictorum, whereas the acid-insoluble matrix accounts for 0.5 %. SDS-PAGE of the ASM reveals the presence of 5 prominent components with apparent molecular weights of 95, 50, 29, 14 and 10 kDa which all stain with CBB (Fig. 1-4). Matrix

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extracted from the two aragonitic layers (prisms + nacre) or from the nacreous layer alone show similar electrophoretical pattern. This demonstrates that all five protein bands are present at least in the nacreous layer. No supplementary major product can be detected with other coloration performed, like the much more sensitive silver nitrate stain (Fig. 2, lanes B). The 95, 50 and 29 kDa bands stain with nitrate silver, CBB, PAS, alcian blue, and ‘Stains-All’ (Fig. 2, lanes B-F). In addition, they all bind Ca45 in vitro (Fig. 2, lane G). These results indicate that the three high molecular weight proteins are acidic and glycosylated, and additionally, bind calcium ions. In the other hand, the 14 and 10 kDa bands observed with CBB, do stain neither with alcian blue, nor with PAS. They do not bind calcium and exhibit a negative staining with silver nitrate and ‘Stains-All’ (not shown). They exhibit biochemical characteristics of acidic proteins associated with biominerals, as reported by Keith et al, 1993.

The 2-D electrophoresis profile reveals that bands observed on 1-D SDS-PAGE gel are composed of numerous isoforms exhibiting various pI (Fig. 3). The 95 kDa-band exhibits 8-9 isoforms, which represent the most acidic pI of the ASM. On the other hand, the 29 kDa-band seems to be composed of 2 distinct protein groups, one represented by a single spot with an acidic pI, the other one composed of four basic spots.

The in vitro crystallization of calcium carbonate shows that the morphology of the crystals is dramatically modified when ASM is added to the medium (Fig. 4). In a blank experiment without any protein added, the crystals obtained are rhombohedric with smooth surfaces. The effect of ASM starts to be apparent at concentrations of 5 μg/ml. At this concentration, the crystals produced have a

Figure 2: SDS-PAGE 12% A) Molecular weight markers (116, 97.4, 65, 45, 31 and 21.5 kDa). B-G) Unio pictorum ASM. A and B: Silver staining, C: Coomassie blue, D: PAS, E: Alcian Blue, F:

‘Stains-All’ and G: Autoradiography with Ca45.

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Figure 3: Two-dimensional gel electrophoresis of the ASM (50 μg load/gel), stained by Coomassie blue (Bio-rad), 3-10 pH and 4-10% polyacrylamide. The first dimension was run with ampholytes.

Figure 4: In vitro crystallisation of calcium carbonate with different concentrations of ASM of Unio pictorum. 1) Blank test. 2) 5 μg/ml. 3) 10 μg/ml. 4) 20 μg/ml (scale bars are respectively 20,

60, 80 and 20 μm).

bigger size. They are characterized by the formation of numerous steps on their sides. By increasing the ASM concentration to 10 μg/ml, the crystals produced are bigger and are all polycrystalline. At higher concentration (20 µg/ml), an inhibiting effect is recorded: the crystals are smaller, less numerous, and exhibit more complex shapes.

The western blot pattern of ASM obtained with an anti-serum raised against P. nobilis nacre matrix (Fig. 5), allowed us to perform a large-scale fractionation

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of the matrix by preparative electrophoresis and to purify the 95 kDa protein. When tested again on a minigel in denaturing conditions, the purified product, provisionally called P95, appears like a single band.

DISCUSSION

The electrophoresis results show the presence of many components in a wide range of molecular weight, in the nacre ASM. In regard to dye patterns, the three high molecular weight products are acidic, densely glycosylated, and bind Ca45 in vitro. The biochemical characterization of these proteins is in progress, in particular, amino acid analysis, N-terminal sequencing, and glycosylation studies. In parallel, because ASM strongly interacts with CaCO3 crystal in vitro, the purified products will be tested individually, to estimate the relative contribution of each of them in the modulation of crystal growth.

Former studies showed the existence of glycoproteins in the nacreous layer of numerous mollusks (Keith et al., 1993, Marxen and Becker, 1997, Sarashina and Endo, 1998, Bédouet et al., 2001, Yin et al., 2005). One can suppose that the contribution of glycans in molluscan shell formation is still widely underestimated. In our future work, we plan to investigate the role of glycosyl moieties in the glycoprotein/crystal interactions (Albeck et al., 1996).

The production of a polyclonal antibody raised against the purified 95 kDa protein of the nacreous ASM is in progress. This will constitute a useful tool for detecting the protein within the shell layers and within the calcifying epithelium. This antibody will also be used for immuno-screening of a cDNA

Figure 5: Purification of the P95 on 12 % SDS-PAGE, silver staining (A, B and C) and Western-blot with anti-nacre of Pinna nobilis (D and E). A) Molecular weight. B and D) ASM. C and E) P95.

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expression library (Marin et al., 2003) constructed from mantle tissues. The new DNA sequences obtained will be compared to those found in pteriomorphid bivalves, for evolutionary purposes.

ACKNOWLEDGEMENTS

This work is supported by an ACI (Action Concertée Incitative, JC 3049) awarded by the French “Ministère délégué à la Recherche et aux Nouvelles Technologies” to F. Marin on the scientific theme “the origin of animal calcification”. B. Marie is recipient from a PhD fellowship of the “Ministère délégué à la Recherche et aux Nouvelles Technologies”. Additional supports are provided by the “Conseil Régional de Bourgogne”.

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