Putative Structure of Acidic Nucleation Sites in Calcified Tissues

One of the critical problems in understanding the mechanisms of matrix-associated mineralization is the lack of information on the three-dimensional structures of biological macromolecules that interface with the mineral. A literature search up to the middle of 1999 yielded very few examples where complete or partial information about the primary structure of macromolecules directly involved in mineralization have been determined (Table 4). Macromolecules isolated from mollusc tissues have traditionally been distinguished into two different classes, based on solubility properties. Chemical analysis showed that the insoluble fraction consists

Figure 5 Fractured surface of the nacreous layer of the bivalve mollusc Atrina rigida. The inset shows the inner nacreous layer of tabular aragonite crystals (top) and the outer prismatic layer of columnar calcite crystals (bottom). SEM micrographs, scale bar denotes 1 ^m. Courtesy of Y. Levi, Department of Structural Biology, Weizmann Institute of Science, Israel.

Figure 5 Fractured surface of the nacreous layer of the bivalve mollusc Atrina rigida. The inset shows the inner nacreous layer of tabular aragonite crystals (top) and the outer prismatic layer of columnar calcite crystals (bottom). SEM micrographs, scale bar denotes 1 ^m. Courtesy of Y. Levi, Department of Structural Biology, Weizmann Institute of Science, Israel.

mainly of fibrous proteins (collagen, chitin) and/or polysaccharides. These macromolecules together build a rigid framework upon which specific macro-molecules from the soluble fraction may become adsorbed. The primary function of the insoluble organic matrix is to subdivide the mineralization compartment into an organized network of microcompart-ments and thus to delimit the available space for crystal growth and/or to constrain the crystal packing arrangement to a certain extent. The surface of this macromolecular assembly may serve as a supramolecular template for oriented nucleation of single crystals, although this structure-function relation is difficult to prove for biological systems in vivo.

The macromolecules contained in the soluble fraction have sequence motifs in common which consist of repeating oligomeric units of acidic residues. Table 4 contains further representative examples of functional macromolecules from calcified tissues. However, the heterogeneity of sources/organisms for isolating these macromolecules, their different

Table 4 Representative structural motifs of macromolecules found in calcified tissues

Name

Source

Repeating oligomeric units

Associated minerai Proposed function

Reference

MSI60

MSI31

MSP-1

Nacrein

Type I collagen

Osteonectin

Pearl oyster: insoluble protein from the nacreous layer

Pearl oyster: insoluble protein from the prismatic layer

Scallop shell: soluble glycoprotein from the foliated shell layer

Pearl oyster: soluble protein from the nacreous layer

Insoluble fibrous scleroprotein from bone

Acidic glycoprotein from bone

Phosphophoryn Soluble highly phosphorylated protein from mineralized dentin

[Ala9_13] and [Gly3_15] Aragonite

[Gly3_5] and Calcite

[/4sp-Gly-Ser-/4sp] and Calcite

[Gly-X-Asn], Aragonite

[Gly-X-Y] triple-helices Hydroxyapatite

[ Giu- Giu-Thr- Giu-Giu- Giu] Hydroxyapatite

(Ser* = phosphorylated serine)

Framework protein Binding of /4sp-rich soluble glycoproteins

Framework protein Binding of Asp-nch soluble glycoproteins

Induction of oriented nucleation

Control of CaC03 polymorphism

Carbonic anhydrase Ca-binding

Framework protein Binding of acidic regulatory proteins

High affinity Ca-binding Collagen binding

Induction of crystal nucleation Regulation of crystal growth Collagen binding

Sarashina and Endo (1998)

Miyamoto et ai. (1996)

Prockop and Feríala (1998)

Fujisawa et ai. (1996)

George et ai. (1996)

PS-2

Acidic polysaccharide from P. carterae coccoliths

COOH COOH

COOH COOH

Calcite

Growth modifier Ca-binding

Marsh (1994)

Sudo S, Fujikawa T, Nagakura T et ai. (1997) Structures of mollusc shell framework proteins. Nature 387: 563-564.

Sarashina I and Endo K (1998) Primary structure of a soluble matrix protein of scallop shell: implications for calcium carbonate biomineralization. American Mineralogist 83: 1510-1515. Miyamoto H, Miyashita T, Okushima M et al. (1996) A carbonic anhydrase from the nacreous layer in oyster pearls. Proceedings of the National Academy of Sciences of the USA 93: 9657-9660.

Prockop DJ and Fertala A (1998) The collagen fibril: the almost crystalline structure Journal of Structural Biology 122: 111-118.

Fujisawa R, Wada Y, Nodasaka Y and Kuboki Y (1996) Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals. Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology 1292: 53-60.

George A, Bannon L, Sabsay B et al. (1996) The carboxyl-terminal domain of phosphophoryn contains unique extended triplet amino acid repeat sequences forming ordered carboxyl-phosphate interaction ridges that may be essential in the biomineralization process. Journal of Biological Chemistry 271, 32869-32873.

Marsh ME (1994) Polyanion-mediated mineralization - assembly and reorganization of acidic polysaccharides in the Golgi system of a coccolithophorid alga during mineral deposition. Protoplasma 177: 108-122.

chemical nature, and their association with different mineral phases clearly rule out a uniform function. They may roughly be divided into five different functional classes:

• concentration regulators: macromolecules that are linked to Ca2+ and/or CO3~ transport and metabolism (e.g. carbonic anhydrase)

• growth inhibitors: acidic macromolecules that strongly bind Ca2+ ions and become nonselective-ly adsorbed on to any arbitrary crystal face which is exposed to the mother liquor

• growth modifiers: acidic macromolecules that interact stereoselectivity with distinct faces of a nascent crystal

• texture modifiers: acidic macromolecules that become occluded and modify texture and mechanic properties of crystals

• nucleators: immobilized acidic macromolecules that form a highly regular template for induced crystal nucleation

Due to the complex nature of interactions in biological matrices, the same acidic macromolecule may belong to more than one of the above mentioned categories and its functional properties may change within different organisms and microenvironments.

To demonstrate a possible mode of molecular interaction between acidic macromolecules and crystal surfaces, this survey of mollusc mineralization will conclude with a brief section about induced CaCO3 nucleation in biological systems. The body fluids of mineralizing organisms contain crystallization inhibitors that prevent spontaneously formed crystal nuclei from growing into larger crystals. To direct mineral deposition to the appropriate location, active nuclea-tion sites have to exist in mineralizing compartments. The opening section about iron storage has already indicated how the molecular architecture of ferritins may be associated with nucleation of iron minerals. For induced calcite and aragonite nucleation, systematic investigations on biological and suitably assembled artificial systems have shed some light on the structural requirements of a putative nucleation site, especially in mollusc shells. The model of Addadi and Weiner proposes structurally pre-organized domains of acidic residues, that could serve as a supramolecu-lar template for oriented crystal nucleation. Such highly ordered domains could result from acidic mac-romolecules being adsorbed on a rigid scaffold of insoluble matrix proteins (Figure 6). As an example, the interlamellar organic sheets of mollusc shell nacre consist of thin sheets of p-chitin (a water-insoluble (1 p 4)-linked 2-acetamido-2-deoxy p-d-glucan) sandwiched between thicker sheets of silk fibroin-like proteins. Silk fibroin itself possesses microcrystalline

Figure 6 Schematic representation of a putative nucleation site in molluscan tissues. An acidic glycoprotein is anchored to a rigid substrate (as schematized by the broken lines) through hydro-phobic or electrostatic interactions. The sulfate groups, linked to flexible oligosaccharide side chains, concentrate Ca2 + ions on an Asp-rich oligopeptide domain that is assumed to adapt a highly regular ^-sheet conformation. A first layer of Ca ions may thus be fixed and oriented in space, upon which further mineral growth ensues. Reproduced with permission from Addadi and Weiner (1989).

Figure 6 Schematic representation of a putative nucleation site in molluscan tissues. An acidic glycoprotein is anchored to a rigid substrate (as schematized by the broken lines) through hydro-phobic or electrostatic interactions. The sulfate groups, linked to flexible oligosaccharide side chains, concentrate Ca2 + ions on an Asp-rich oligopeptide domain that is assumed to adapt a highly regular ^-sheet conformation. A first layer of Ca ions may thus be fixed and oriented in space, upon which further mineral growth ensues. Reproduced with permission from Addadi and Weiner (1989).

domains of repeating [Gly-Ala-Gly-Ala-Gly-Ser]n units that adopt an antiparallel ^-pleated sheet conformation. These domains have a highly regular and hydrophobic surface upon which acidic macro-molecules are adsorbed from solution. In the course of adsorption, the acidic macromolecules has to fold into the appropriate conformation, in order to maximize its hydrophobic interactions with the silk fibroin surface. Possible candidates for acidic macromolecules interacting with silk fibroin in the described way are oligopeptides that include sequence motifs of [Asp-X]n, (X = Gly, Ser), which have a strong tendency to fold into a ^-sheet conformation in the presence of Ca2+ ions. As a consequence, the aspartic acid residues of [Asp-X]n sequences would be positioned at only one side of the ^-pleated sheet, resulting in an organized two-dimensional array of carboxylate ligands.

It is tempting to assume that the carboxylate residues coordinate a first layer of Ca2+ ions which would in turn become the first layer of an epitaxially growing CaCO3 crystal. However, a more profound analysis has so far failed to provide evidence for an epitaxial growth mechanism or a close stereochem-ical complementarity between the nucleating macro-molecules and the incipient CaCO3 crystal surface. Some properties of the mollusc shell ultrastructure rather point to less sophisticated nucleation strategies; examination of the common crystal orientations in a variety of calcifying organisms reveal that aragonite and calcite single crystals most frequently nucleate from the ab planes. The arrangement of Ca2 + ions in this plane (the shortest distance between Ca2 + ions is 4.99 A in calcite, and 4.69 A in aragonite, respectively; Figure 3) is geometrically not commensurate with the period of amino acid residues in a protein ft-strand (approx. 6.9 A). Moreover, on pointing more or less perpendicular towards the Ca2 + ions in the crystal (0 0 1) face, the carboxylate residues of the ft-pleated sheet cannot continue the parallel arrangement of planar carbonate anions in the underlying layer(s). The current nucleation model thus does not support the picture of a calcite or aragonite single crystal being nucleated from (0 0 1) crystal faces by virtue of stereochemical selection principles.

Despite the similar positioning of Ca2 + ions in the ab plane of calcite and aragonite, mollusc shells discriminate between the two polymorphs by secreting them separately in different layers (e.g. prismatic layer and nacre). This suggests that additional factors participate in nucleation. One possibility is that different Mg2 + concentrations in the fluids of aragonitic and calcitic layers may be present that could help to shift the balance between the two polymorphs. Another possibility is the presence of polymorph-specific macromolecules that interact with more than one face of the nascent crystal. For a valid explanation of selective nucleation of either polymorph, the current, essentially geometric model will have to be refined. A lot is expected from the first three-dimensional structure of a nucleating macromolecule, although its active conformation may depend on the accompanying insoluble organic matrix in the biological tissues. Finally, novel theoretical approaches are currently being investigated to explore realistic surface properties of the CaCO3 polymorphs which consider surface relaxation as well as hydration of the outermost ionic layers.

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