The interstitial crystal-nucleating sheet in molluscan Haliotis rufescens shell: A bio-polymeric composite

In honor of Professor Alberto Ripamonti in the occasion of the 80th birthday.
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Abstract

The interstitial green sheets in abalone shell nacre are shown to be bifacially differentiated trilaminate polymeric complexes, with glycoprotein layers sandwiching a central core containing chitin. They share some common feature with the organic matrix layers between the aragonite tablets in the nacre and the periostracum, and show similarities to the myostracum. Thus, although the green sheet is reported to be unique to the abalone shell, it represents an interesting model for the study of molluscan shell biomineralization processes. Indeed, during shell formation, prismatic and spherulitic aragonite precedes and follows the deposition of the interstitial green polymeric composite sheets, and there is evidence to suggest that these sheets demark the interruption of nacre synthesis and serve to nucleate the resumption of calcium carbonate crystal growth. The green polymeric interstitial sheet purified from the abalone shell was investigated by spectroscopic and imaging techniques: FTIR, confocal microscopy, scanning and transmission electron microscopy, and by pyrolysis combined with GC–MS. Structural and compositional differences are observed between the surfaces of the two sides of the interstitial polymeric composite sheets. Moreover, comparative crystallization experiments on the green sheet sides also reveal asymmetry with respect to the nucleation of calcium carbonate. These findings suggest that these bifacially differentiated interstitial composites may play an active role in the mineral assembly processes, with one of the surfaces acting as a crystal nucleator.

Introduction

The shell of the of the gastropod mollusc, Haliotis rufescens (red abalone), is a natural high-performance micro-laminate composite of inorganic crystals and biopolymers with a fracture toughness estimated to be 3000-times greater than that of the constituent mineral alone, although the content of organics represents only ca 1% of the mass of the composite (Belcher et al., 1998). The macrostructure of the shell consists of layers of calcite and aragonite, each associated with a characteristic family of biopolymers. In the order of their sequential deposition these include a red prismatic calcite layer (that forms the outer layer of the shell) and an iridescent, inner, aragonitic layer called nacre. Additional interstitial heterolayers consisting of non-nacreous mineral and green organic sheets are interspersed through the nacre at irregular intervals (Erasmus et al., 1994, Shepherd et al., 1995, Hawkes et al., 1996, Zaremba et al., 1996, Su et al., 2002). A temporal and spatial sequence of layered growth resembling that of the shell also is observed on abiotic substrates inserted between the growing surface of the shell and the mantle (the secretory epithelium), producing a flat pearl (Fritz et al., 1994, Belcher et al., 1996, Su et al., 2002). Dissolution of the calcium carbonate of the nacreous portions of the abalone shell or flat pearl leaves an insoluble fraction representing the bulk of the organic matrices. This heterogeneous mixture of biopolymers includes a matrix of sheets from the nacre and the green polymeric composite sheets from the interstitial heterolayers (Zaremba et al., 1996, Belcher et al., 1996, Su et al., 2002).

Because the green polymeric sheets exhibit structural and functional similarities to the nucleating protein sheet that is first deposited on abiotic substrates implanted adjacent to the mantle, Belcher et al. (1996) used the purified interstitial green sheets to nucleate CaCO3 crystallization, demonstrating: (a) that the polymer composite sheets from the heterolayers exhibit nuclein-like activity; and (b) that the addition of soluble proteins extracted from the calcitic and aragonitic domains of the shell can control the resulting crystal polymorph and atomic lattice orientation of the growing crystals. The interstitial nucleating sheet was known to be colored (green) and autofluorescent; it stains with both Coomassie brilliant blue (indicating that it contains protein) and with a cationic carbocyanin dye (“Stains-All”, indicating that it contains high local concentrations of anionic groups), and it is resistant to a wide range of denaturing agents and proteolytic enzymes, indicating a high degree of covalent cross-linking. Little else had been known about the composition and structure of this polymeric composite. Only recently specific proteoglycans have been localized in the green sheet or meso layer (Fernandez et al., 2007, Fernandez and Arias, 2008).

The shell of molluscs has, as its outermost layer, a coating called the periostracum, providing it with some protection from the environment (Clark, 1976). This also provides the substrate upon which molluscan shell mineralization begins (Iwata, 1980). Proteins are the major constituent of the periostracum, with chitin variably present as a minor constituent (Goffinet and Jeuniaux, 1979). In the periostracum the macromolecules are highly polymerized by a process that involves DOPA-(3,4-dihoxyphenylalanine-)containing protein in bivalves, and probably involves dityrosine residues in gastropods, analogously to insect cuticle (Waite, 1983a). The periostracum generally shows little or no ultrastructural order at the molecular level, although subdivision of various layers has been described (Kniprath, 1972, Waite, 1977, Saleuddin and Petit, 1983, Harper, 1997).

An additional distinct layer observed in mollusc shells is described as the myostracum, formed at the site of attachment of muscles to the shell. The myostracum is always formed of aragonite, uniquely exhibiting prismatic ultrastructure (Bøggild, 1930). Only one acidic protein from the myostracum has thus far been purified and sequenced (Lee et al., 2006).

The nature and properties of the organic polymer matrix of nacre has been most extensively characterized. The complete sequence of lustrin A, a modular protein with multiple repeating, alternating cysteine-rich and proline-rich domains suspected of functioning as an elastomeric linker, has been determined from its cloned cDNA (Shen et al., 1997). Since this discovery, a number of matrix proteins has been identified from abalone nacre. These are comprised of C-type lectin perlucin, which promotes CaCO3 crystal nucleation in vitro (Mann et al., 2000); perlustrin, a small growth factor-binding protein (Weiss et al., 2001); perlwapin, a protein containing three whey-acidic protein domains (Treccani et al., 2006) and perlinhibin, a cysteine-, histidine-, and arginine-rich protein which inhibits in vitro calcium carbonate crystallization (Mann et al., 2007). However, none of these was able to specifically control aragonite deposition. Only recently the complete sequence of a protein from nacre able to induce aragonite precipitation, Pif, has been obtained (Suzuki et al., 2009).

Weiner and Traub showed by electron and X-ray diffraction that the layers between successive aragonite tablets in the nacre of bivalve molluscs include protein with beta-sheet structure resembling that of silk fibroin and polysaccharide resembling beta-chitin (Weiner and Traub, 1980, Weiner et al., 1983). Electron microscopy had suggested that the intercrystalline polymer sheets in abalone nacre are organized with the chitin layer sandwiched between two layers of protein (Mutvei, 1969, Nakahara et al., 1982). Falini et al. (1996) used the information from the Weiner and Traub research (Weiner and Traub, 1980, Weiner et al., 1983) to develop a model system in which soluble glycoproteins control the polymorph of calcium carbonate crystals nucleated in a heterologous composite substrate formed from purified squid chitin and silk fibroin. Electron cryo-microscopy of nacre matrices revealed that chitin and silk fibroin-like proteins are assembled into alternating layer-by-layer structure of species-specific finite thickness (Nakahara et al., 1982). Further studies have shown that the fibroin-like proteins form a gelling environment in which the growth of the mineral phase takes place (Levi-Kalisman et al., 2001). This result was subsequently supported by the preparation of polymeric blends of silk fibroin and chitin (Falini et al., 2003). Recently it has been demonstrated that chitin also acts as a chemical substrate to which glycoproteins are bound covalently (Weiss et al., 2009).

We present here a characterization of the green polymeric interstitial sheet purified from the abalone shell. This material is composed principally of protein and polysaccharide in an asymmetric, bifacially differentiated micro-laminate with a chitin-containing core. The structure of the sheet resembles in some aspects that of the polymeric matrix associated with the nacre, which also consists of a layered structure of proteins surrounding an internal core of β-chitin. The proteinaceous layers of the interstitial polymer composite sheets are uniquely tyrosine-rich and highly cross-linked via tyrosine side-chains. This characteristic has been observed in the periostracum of shells (Meenakshi et al., 1971, Waite, 1977, Waite, 1983a, Zhang et al., 2006). Some of the constituent proteins can be extracted in soluble form with a chaotropic buffer and a peptide has been isolated after alkaline digestion. The calcium carbonate nucleating properties of the green sheet sides have been investigated as well.

Section snippets

Purification of the interstitial polymer composite sheets

Shells of red abalones (Haliotis rufescens) were obtained from live specimens (collected at Santa Barbara, CA), air-dried and stored. Portions of the shell corresponding to the outer prismatic calcite and inner nacreous domains were mechanically separated, cleaned, and decalcified in dilute acetic acid containing 0.1% sodium azide (with or without 0.25% v/v glutaraldehyde as fixative, as specified) at 20 °C, and the pH maintained at 3.5. After decalcification, salts were removed from the

Results

The green polymeric sheets are interspersed throughout the nacre at irregular intervals, sandwiched between two layers of aragonite that differ in morphology from the tablet-like crystals composing the surrounding nacre. Proceeding in the order of growth-deposition, an aragonitic prismatic layer less than 10 μm thick is deposited on the nacre before the green sheet is deposited, while on the green sheet, subsequent crystallization of spherulites occurs, formed by the aggregation of acicular

Discussion

The green polymeric heterolayer is a unique characteristic, to our knowledge, of the abalone shell (Erasmus et al., 1994); this fact apparently may reduce its general importance in the understanding of molluscan shell biomineralization processes. Moreover, the green sheet may be produced in response to external perturbations in the growth of nacre, as it is deposited at irregular intervals in the shell (Hawkes et al., 1996). However, the significance of the green sheet can be appreciated when

Conclusion

The interstitial green sheets in abalone shell nacre are trilaminate, with glycoprotein layers sandwiching a central core containing chitin. In this respect, the architecture of the interstitial sheets resembles that of the insoluble organic matrix of the nacre, although the proteins are very different in the two materials. The composition of the green sheet is similar to that of the periostracum. In these composites a high content of tyrosine and its derivatives is present; these residues are

Acknowledgments

G.F. thank Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRC MSB), the University of Bologna (Funds for Selected Topics) and Ministero dell’Istruzione dell’Università e della Ricerca for the financial support. Work at UCSB was supported by a MURI grant from the ARO. We thank Dr. Erik Caroselli for the help in the statistical analyses.

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