Promotion of fluorapatite crystallization by soluble-matrix proteins from Lingula anatina shells.

Abstract

One of the most persistent enigmas in the field of biomineralization is the evolutionary selection of calcium carbonate and calcium phosphate biominerals in invertebrate and vertebrate skeletal tissues, respectively. Whereas bones and teeth are constructed with carbonate-substituted hydroxyapatite (Ca10(PO4)6(OH)2), most mollusc shells consist of polymorphs of calcium carbonate such as calcite and aragonite. One of the few modern exceptions to this carbonate–phosphate dichotomy in skeletal biomineralization is the bivalved shell of extant Linguliform brachiopods such as Lingula and Discinisca, which consists of carbonate-substituted fluorapatite, (Ca10(PO4)6F2, francolite, FAP). [2] Lingula shells appeared along with phosphatic biominerals in the Problematica at the base of the Cambrian, about 545 million years ago (Mya), possibly because of high concentrations of phosphorus in the seawater during the Precambrian and Cambrian eras (543 to 490 Mya). The subsequent decrease in phosphate levels by the end of the Cambrian era may account for the extinction of the phosphatic Problematica due to arrested shell mineralization, although a switch to a carbonate regime may also have occurred in these invertebrates during this era. The geological longevity of Lingula, in contrast, suggests that the lightly mineralized FAP shell has remained well adapted to evolutionary pressures even though by the middle of the Ordovician (ca. 470 Mya) phosphatic biomineralization was rare in invertebrates. Previous studies have shown that the Lingula shell is produced by a rhythmic alternation of organic and mineralized layers. 5] The latter are constructed from FAP granules, 4–8 nm in diameter, which are smaller at the anterior and periphery of the shell compared with those deposited in the central and posterior regions, and become progressively aligned with the organic matrix during mineralization. The matrix consists of a complex mixture of glycosaminoglycans, b-chitin, and proteins with molecular masses between 6 kDa to 46 kDa, many of which are enriched in acidic amino-acid residues. 11] Because acidic macromolecules are considered in general to play a pivotal role in the nucleation and growth of calcium-containing biominerals, we have investigated the potential role of Lingula shell proteins on the formation of carbonate-substituted FAP crystals in vitro. Herein we show for the first time that soluble macromolecules isolated from Lingula shells specifically promote FAP crystallization by the destabilization of an amorphous calcium phosphate precursor. Proteins were sequentially extracted from powdered L. anatina whole valves or from four equally sized sections from the posterior to the anterior of the shell. Fractionation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS PAGE) gave discrete bands that correspond to proteins with estimated molecular masses of 6, 8.5, 15.5, 21.5, 24, and 44 kDa for guanadine hydrochloride (GnHCl) and EDTA extracts (Figure 1A). The EDTA extract contained significantly higher concentrations of the lower-molecular-weight proteins as well as additional proteins of molecular mass 4.5, 28, 35, 40, 50, 57.5, 60, and 69 kDa. The 21.5 and 24 kDa proteins in both extracts were glycosylated as determined by using concanavalin A binding (Figure 1B,C). The influence of Lingula shell proteins on the in vitro deposition of carbonate-substituted FAP was investigated by adding mixtures of the extracted macromolecules at various total concentrations to buffered calcium phosphate/fluoride metastable solutions at constant temperature. In each case, metastability was induced by raising the solution pH from 6.2 to 7.5, which resulted in relatively slow growth kinetics that were monitored by a progressive reduction in solution pH with time due to deprotonation of [H2PO4] and [HPO4] 2