A transporter that allows phosphate ions to control the polymorph of exoskeletal calcium carbonate biomineralization.

The SLC20A2 transporter supplies phosphate ions (Pi) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of Pi are found in the CaCO3-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and Pi transport to exoskeletal elements was studied with respect to the role of Pi in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for Pi transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by Pi. RNA interference experiments via CqSLC20A2 dsRNA injections reduced Pi content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO3) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced Pi content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which Pi affects CaCO3 biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of Pi as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: • Crustaceans exoskeletons are hardened mainly by CaCO3, with Pi in minute amounts • Pi was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo • For the first time, transport protein for Pi was discovered in crayfish • Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced Pi.

Genes encoding putative bicarbonate transporters as a missing molecular link between molt and mineralization in crustaceans.

During their life, crustaceans undergo several molts, which if theoretically compared to the human body would be equivalent to replacing all bones at a single event. Such a dramatic repetitive event is coupled to unique molecular mechanisms of mineralization so far mostly unknown. Unlike human bone mineralized with calcium phosphate, the crustacean exoskeleton is mineralized mainly by calcium carbonate. Crustacean growth thus necessitates well-timed mobilization of bicarbonate to specific extracellular sites of biomineralization at distinct molt cycle stages. Here, by looking at the crayfish Cherax quadricarinatus at different molting stages, we suggest that the mechanisms of bicarbonate ion transport for mineralization in crustaceans involve the SLC4 family of transporters and that these proteins play a key role in the tight coupling between molt cycle events and mineral deposition. This discovery of putative bicarbonate transporters in a pancrustacean with functional genomic evidence from genes encoding the SLC4 family-mostly known for their role in pH control-is discussed in the context of the evolution of calcium carbonate biomineralization.

MARS: A protein family involved in the formation of vertical skeletal elements.

Vertical organizations of skeletal elements are found in various vertebrate teeth and invertebrate exoskeletons. The molecular mechanism behind the development of such structural organizations is poorly known, although it is generally held that organic matrix proteins play an essential role. While most crustacean cuticular organizations exhibit horizontal chitinous layering, a typical vertical organization is found towards the surface of the teeth in the mandibles of the crayfish Cherax quadricarinatus. Candidate genes encoding for mandible-forming structural proteins were mined in C. quadricarinatus molt-related transcriptomic libraries by using a binary patterning approach. A new protein family, termed the Mandible Alanine Rich Structural (MARS) protein family, with a modular sequence design predicted to form fibers, was found. Investigations of spatial and temporal expression of the different MARS genes suggested specific expression in the mandibular teeth-forming epithelium, particularly during the formation of the chitinous vertical organization. MARS loss-of-function RNAi experiments resulted in the collapse of the organization of the chitin fibers oriented vertically to the surface of the crayfish mandibular incisor tooth. A general search of transcriptomic libraries suggested conservation of MARS proteins across a wide array of crustaceans. Our results provide a first look into the molecular mechanism used to build the complex crustacean mandible and into the specialized vertical structural solution that has evolved in skeletal elements.

Calcium phosphate mineralization is widely applied in crustacean mandibles.

Crustaceans, like most mineralized invertebrates, adopted calcium carbonate mineralization for bulk skeleton reinforcement. Here, we show that a major part of the crustacean class Malacostraca (which includes lobsters, crayfishes, prawns and shrimps) shifted toward the formation of calcium phosphate as the main mineral at specified locations of the mandibular teeth. In these structures, calcium phosphate is not merely co-precipitated with the bulk calcium carbonate but rather creates specialized structures in which a layer of calcium phosphate, frequently in the form of crystalline fluorapatite, is mounted over a calcareous "jaw". From a functional perspective, the co-existence of carbonate and phosphate mineralization demonstrates a biomineralization system that provides a versatile route to control the physico-chemical properties of skeletal elements. This system enables the deposition of amorphous calcium carbonate, amorphous calcium phosphate, calcite and apatite at various skeletal locations, as well as combinations of these minerals, to form graded composites materials. This study demonstrates the widespread occurrence of the dual mineralization strategy in the Malacostraca, suggesting that in terms of evolution, this feature of phosphatic teeth did not evolve independently in the different groups but rather represents an early common trait.

A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix properties.

Some crustaceans possess exoskeletons that are reinforced with calcium carbonate. In the crayfish Cherax quadricarinatus, the molar tooth, which is part of the mandibular exoskeleton, contains an unusual crystalline enamel-like apatite layer. As this layer resembles vertebrate enamel in composition and function, it offers an interesting example of convergent evolution. Unlike other parts of the crayfish exoskeleton, which is periodically shed and regenerated during the molt cycle, molar mineral deposition takes place during the pre-molt stage. The molar mineral composition transforms continuously from fluorapatite through amorphous calcium phosphate to amorphous calcium carbonate and is mounted on chitin. The process of crayfish molar formation is entirely extracellular and presumably controlled by proteins, lipids, polysaccharides, low-molecular weight molecules and calcium salts. We have identified a novel molar protein termed Cq-M15 from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. Its transcript and differential expression were confirmed by a next-generation sequencing library. The predicted acidic pI of Cq-M15 suggests its possible involvement in mineral arrangement. Cq-M15 is expressed in several exoskeletal tissues at pre-molt and its silencing is lethal. Like other arthropod cuticular proteins, Cq-M15 possesses a chitin-binding Rebers-Riddiford domain, with a recombinant version of the protein found to bind chitin. Cq-M15 was also found to interact with calcium ions in a concentration-dependent manner. This latter property might make Cq-M15 useful for bone and dental regenerative efforts. We suggest that, in the molar tooth, this protein might be involved in calcium phosphate and/or carbonate precipitation.

A novel chitin binding crayfish molar tooth protein with elasticity properties.

The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.

Layered growth of crayfish gastrolith: about the stability of amorphous calcium carbonate and role of additives.

Previous studies on pre-molt gastroliths have shown a typical onion-like morphology of layers of amorphous mineral (mostly calcium carbonate) and chitin, resulting from the continuous deposition and densification of amorphous mineral spheres on a chitin-matrix during time. To investigate the consequences of this layered growth on the local structure and composition of the gastrolith, we performed spatially-resolved Raman, X-ray and SEM-EDS analysis on complete pre-molt gastrolith cross-sections. Results show that especially the abundance of inorganic phosphate, phosphoenolpyruvate (PEP)/citrate and proteins is not uniform throughout the organ but changes from layer to layer. Based on these results we can conclude that ACC stabilization in the gastrolith takes place by more than one compound and not by only one of these additives.

Enamel-like apatite crown covering amorphous mineral in a crayfish mandible.

Carbonated hydroxyapatite is the mineral found in vertebrate bones and teeth, whereas invertebrates utilize calcium carbonate in their mineralized organs. In particular, stable amorphous calcium carbonate is found in many crustaceans. Here we report on an unusual, crystalline enamel-like apatite layer found in the mandibles of the arthropod Cherax quadricarinatus (freshwater crayfish). Despite their very different thermodynamic stabilities, amorphous calcium carbonate, amorphous calcium phosphate, calcite and fluorapatite coexist in well-defined functional layers in close proximity within the mandible. The softer amorphous minerals are found primarily in the bulk of the mandible whereas apatite, the harder and less soluble mineral, forms a wear-resistant, enamel-like coating of the molar tooth. Our findings suggest a unique case of convergent evolution, where similar functional challenges of mastication led to independent developments of structurally and mechanically similar, apatite-based layers in the teeth of genetically remote phyla: vertebrates and crustaceans.

Stabilization of amorphous calcium carbonate by phosphate rich organic matrix proteins and by single phosphoamino acids.

Stable amorphous calcium carbonate (ACC) is a unique material produced naturally exclusively as a biomineral. It was demonstrated that proteins extracted from biogenic stable ACC induce and stabilize synthetic ACC in vitro. Polyphosphate molecules were similarly shown to induce amorphous calcium carbonate formation in vitro. Accordingly, we tested the hypothesis that biogenic ACC induction and stabilization is mediated by the phosphorylated residues of phosphoproteins. We show that extracellular organic matrix extracted from gastroliths of the red claw crayfish Cherax quadricarinatus induce stable ACC formation in vitro. The proteinaceous fraction of this organic matrix is highly phosphorylated and is incorporated into the ACC mineral phase during precipitation. We have identified the major phosphoproteins of the organic matrix and showed that they have high calcium binding capacity. Based on the above, in vitro precipitation experiments with single phosphoamino acids were performed, indicating that phosphoserine or phosphothreonine alone can induce the formation of highly stable ACC. The results indicate that phosphoproteins may play a major role in the control of ACC formation and stabilization and that their phosphoamino acid moieties are key components in this process.

A protein involved in the assembly of an extracellular calcium storage matrix.

Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.

The role of seawater endocytosis in the biomineralization process in calcareous foraminifera.

Foraminifera are unicellular organisms that inhabit the oceans in various ecosystems. The majority of the foraminifera precipitate calcitic shells and are among the major CaCO(3) producers in the oceans. They comprise an important component of the global carbon cycle and also provide valuable paleoceanographic information based on the relative abundance of stable isotopes and trace elements (proxies) in their shells. Understanding the biomineralization processes in foraminifera is important for predicting their calcification response to ocean acidification and for reliable interpretation of the paleoceanographic proxies. Most models of biomineralization invoke the involvement of membrane ion transporters (channels and pumps) in the delivery of Ca(2+) and other ions to the calcification site. Here we show, in contrast, that in the benthic foraminiferan Amphistegina lobifera, (a shallow water species), transport of seawater via fluid phase endocytosis may account for most of the ions supplied to the calcification site. During their intracellular passage the seawater vacuoles undergo alkalization that elevates the CO(3)(2-) concentration and further enhances their calcifying potential. This mechanism of biomineralization may explain why many calcareous foraminifera can be good recorders of paleoceanographic conditions. It may also explain the sensitivity to ocean acidification that was observed in several planktonic and benthic species.

A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix.

Despite the proclamation of Lowenstam and Weiner that crustaceans are the "champions of mineral mobilization and deposition of the animal kingdom," relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization.