Biomineralisation of the Calcitic-Shelled, Inarticulated Brachiopod, Neocrania anomala

The inarticulated calcitic shelled brachiopod, Neocrania anomala belongs to the craniids, the sole Recent members of the class Craniata within the subphylum Craniiformea. Articulation is associated with calcitic valves in the Rhynchonelliformea and inarticulation with apatitic valves in the Linguliformea. Biominerals have organic material, including proteins and glycoproteins, intimately associated with the mineral phase, reducing the nucleation energy required for mineral precipitation and influencing the formation of the biomineral. The characteristics of the structure of shell are therefore a consequence of the protein component of the constituent biomineral. The intracrystalline proteins of N. anoniala are examined here to gain information relating to the formation of the biomineral and therefore the shell of N. anomala. The intracrystalline protein fraction of the shell of N. anomala, is here defined as the protein which is resistant to strong chemical oxidation by incubation of powdered shells in an aqueous solution of sodium hypochlorite (1% v/v) for one hour. Mineral-associated proteins were extracted from the intracrystalline and intercrystalline fractions of the dorsal valve and the intracrystalline fraction of the ventral valve. The proteins were purified to homogeneity using gel electrophoresis (SDS PAGE) and subsequently analysed by partial N-terminal sequencing and amino acid analysis. A 44 kDa protein is present in both the intracrystalline and total protein extracts of the dorsal valve and also in the intracrystalline fraction of the ventral valve. In addition, the intracrystalline fraction of the dorsal valve contains a 60 kDa protein. The partial N-terminal sequence obtained for the 44 kDa protein does not resemble previously sequenced brachiopod proteins, nor any other protein in the protein databases searched. The dorsal valve has a higher concentration of protein than the ventral valve. Differences exist between the two valves in terms of the amount of protein present in different locations within the shell. The dorsal valve has twice as much intracrystalline protein per weight of shell than the ventral valve. The higher proportion of protein in the dorsal valve may be related to the presence of seminacre in this valve. The 44 kDa protein of the intracrystalline fraction of the dorsal valve has high levels of aspartic acid/asparagine and glutamic acid/glutamine, as well as glycine and serine. The EDTA-insoluble material contains significantly more protein than the EDTA-soluble protein extract from the intracrystalline fraction of the dorsal valve and differences exist between the two extracts in terms of amino acid composition. The soluble protein of the dorsal valve can both initiate and inhibit crystal nucleation in vitro, depending on the concentration of the protein in the system. Whole extracts were added to the system, so it is not possible to identify which protein(s) influences crystal nucleation. The 44 kDa and the 60 kDa proteins both possess a calcium-binding ability, as determined by reaction with Stains-all and this may be suggestive of the proteins involvement in biomineralisation in vivo. The 60 kDa protein is glycosylated, whereas the 44 kDa protein is not. Chemical etching of the laminar calcite that comprises the semi-nacreous secondary layer of the dorsal valve of N. anornala reveals a high order structural arrangement that resembles the ultrastructures observed in nacreous bivalve shells. Nucleation of new crystals occurs uniformly over the surface of the individual tablets, an arrangement which is also observed in the semi-nacre of the cyclostome bryozoans. The non-specific nucleation observed in these groups may be related to the formation of semi-nacreous shell layers. The presence and pattern of the ultrastructures of the N. anomala secondary shell suggests that lophophorates possess a more complex system of biomineralisation than the Lophophorate-Mollusc discontinuity concept suggests