Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein

Abstract

Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called "calcite and aragonite seas." Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type nearfield IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that -OH, secondary structures (e.g. ␣-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature.The primary mineralogy of marine carbonates has changed over geological history depending on the magnesium/calcium ratio in seawater, including during the so-called "aragonite sea" (1) period and two periods of "calcite seas" (2). This ratio is apparently driven by changes in spreading rates along midocean ridges (1). The calcification process of aragonite and calcite mineralogy in mollusk shells (3-8) and some information about calcite (9, 10) have been reported, but our knowledge of the mechanism of the direct biological formation of calcite in marine organisms, especially in corals, remains incomplete. To develop a more complete understanding of calcite formation, detailed information concerning how biomolecules contribute to the kinetics of crystal formation, as well as the structural details of both the surface and the interior of single crystals in the submicrometer to nanometer scale must be analyzed. The mechanism of calcite formation in soft coral sclerites that we report here is completely different from the mechanisms used by other calcifying marine organisms, featuring new chemical groups and different types of single crys...

Analysis of the Proteinaceous Components of the Organic Matrix of Calcitic Sclerites from the Soft Coral Sinularia sp.

“…The tryptic peptides were separated by a nano-LC and analyzed by MS/MS using a highly powerful MALDI-TOF-TOF mass spectrometer. Although marine organisms have been reported to contain carbonic anhydrase [8], [9], [10], [11], [12], calcium-binding and glycosylated proteins [1], [10], [13], [14], this is the first report of the identification of actin in sclerites. Actin is a protein that has not previously been shown to play a role in the biocalcification process of other corals except in the planular larvae of the scleractinian Galaxea fascicularis [15], but it has been suggested that actins reach the area of biomineralization as a by-product of the secretion of other proteins involved in calcification and may not be directly involved in the biomineralization process [17].…”

“…Recently, actin was identified in the intracellular organic matrix of sea urchin tests and spines [17] and in the planular larvae of scleractinian corals [15] but its direct involvement in the biocalcification process has yet to be demonstrated. Here, we found that actin proteins in the sclerites of Sinularia sp., which also contain the mineral calcite [14], [18], are secreted together with an organic matrix and subsequently transported to the outside of the cell where extracellular calcification occurs. This process is similar to sclerite calcification in other octocorallians [19], [20].…”

“…Mature sclerites are completely free of cellular materials and ultimately become extracellular structures. We are tempted to exclude actin contamination from the surrounding cells as an explanation for this finding due to the many peptide fragments identified as actin from a complex protein mixture that was purified from silver-stained protein bands using our newly established matrix protein purification technique [1], [14]. However, verification of these proteins using a number of techniques (e.g., CBB staining, western blotting, silver staining, and analyses by nanoLC-MS/MS using a MALDI-TOF-TOF) was done several times on the same protein bands with the same results.…”

Intra-skeletal calcite in a live-collected Porites sp.: Impact on environmental proxies and potential formation process

“…6) we calculated a valueof 17.05±0.85‰ for a virtual 100%-calcite end-memberwith a constant seawater δ 11 B value of 39.61‰ .This would correspond to a calculatedpH solution of about 7.87 ± 0.11. It can be noted that such low δ 11 B value was recently observed for calcite cold-water octocorals (Farmer et al, 2015;McCulloch et al, 2012) which calcifying fluid pH fallingwithin 7.6-8.0.Nevertheless, in a biologically-controlled biomineralization, aragonite and calcite precipitation strictly results from the control of the organic matrix compounds,in the scleractinian coral case synthesized by the calicoblastic epithelium (Falini et al, 1996;Falini et al, 2013;Mass et al, 2013;Puverel et al, 2005;Rahman et al, 2011;. To date, no coral specieshasbeen found to produce both CaCO 3 polymorphsin natural modern seawater conditions, even in the primary skeleton formed by coral post-larvae (Clode et al, 2011).…”

Bioactive Cembranoid Composition in the Soft Coral of Sarcophyton glaccum on The Response to Changing pH

“…The higher calcite saturation in natural and acidification environments is an advantage to soft coral species. Different from hard coral that uses aragonite, soft coral has the ability to use calcite for its skeleton (Rahman et al, 2011). Therefore, the natural composition of carbonate chemistry under increased ocean acidification conditions is definitely a factor influencing the resilience of soft corals in such conditions.…”