In situ molecular NMR picture of bioavailable calcium stabilized as amorphous CaCO₃biomineral in crayfish gastroliths

Abstract

Bioavailable calcium is maintained by some crustaceans, in particular freshwater crayfish, by stabilizing amorphous calcium carbonate (ACC) within reservoir organs—gastroliths, readily providing the Ca 2+ needed to build a new exoskeleton. Despite the key scientific and biomedical importance of the in situ molecular-level picture of biogenic ACC and its stabilization in a bioavailable form, its description has eluded efforts to date. Herein, using multinuclear NMR, we accomplish in situ molecular-level characterization of ACC within intact gastroliths of the crayfish Cherax quadricarinatus . In addition to the known CaCO 3 , chitin scaffold and inorganic phosphate (Pi), we identify within the gastrolith two primary metabolites, citrate and phosphoenolpyruvate (PEP) and quantify their abundance by applying solution NMR techniques to the gastrolith “soluble matrix.” The long-standing question on the physico-chemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state rotational-echo double-resonance (REDOR) and transferred-echo double-resonance (TEDOR) NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found < 5 Å from a phosphate (intermolecular C⋯P distance), an interaction that must be mediated by Ca 2+ . The high abundance and extensive interactions of these molecules with the ACC matrix identify them as the central constituents stabilizing the bioavailable form of calcium. This study further emphasizes that it is imperative to characterize the intact biogenic CaCO 3 . Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure, and molecular functionality in situ.

Interplay between Calcite, Amorphous Calcium Carbonate, and Intracrystalline Organics in Sea Urchin Skeletal Elements

“…This confirms the presence of ACC domains in both samples, as already proposed in sea urchin spines. 47 The FWHM of the ACC 13 C resonance from sea urchin skeletal elements is twice smaller than the one reported for synthetic ACC 58,59 and biogenic ACC gastrolith (enriched in P) 60,61 , suggesting that ACC in mature sea urchin large spines and test plates is more ordered than stable biogenic ACC in crayfish 60 and lobster 61 gastrolith. The relatively low organics content determined by TGA (about 1.4 wt.…”

“…Compared to synthetic ACC and other long-living biogenic ACC (gastrolith), 58,60 we showed that ACC in sea urchins is less disordered and that the low water content is most likely bound to the ACC phase (Ca:H 2 O 1:0.1). Therefore, the ACC in sea urchin skeletal elements is relatively anhydrous in comparison with other long-living biogenic ACCs (e.g.…”

Representing calcification in distribution models for aquatic invasive species: surrogates perform as well as CaCO3 saturation state

“…Both calcium and bicarbonate are at least partially supplied from ambient water, although some organisms may internally store CaCO 3 to be used when needed (Horne et al, 2009;Akiva-Tal et al, 2011;Marin et al, 2012). Low concentrations of dissolved calcium can impose limits on CaCO 3 mineralization, so dissolved calcium and correlated parameters are used in biological models.…”

High-resolution structural and elemental analyses of calcium storage structures synthesized by the noble crayfish Astacus astacus

“…Phosphorylated proteins have been characterized and hypothesized as playing a role in Cherax gastrolith ACC stabilization Glazer and Sagi, 2012;Glazer et al, 2010;Shechter et al, 2008b). Small phosphorylated metabolites of the glycolytic pathway have also been evidenced by Nuclear Magnetic Resonance (NMR) and thought to play an interacting role between matrix proteins and calcium ions forming complexes preventing the crystallization of CaCO 3 (Akiva-Tal et al, 2011;Sato et al, 2011).…”

“…Akiva-Tal et al, 2011;Bentov et al, 2010;Habraken et al, 2015;Luquet et al, 2016;Reeder et al, 2013;Reid et al, 2012;Sato et al, 2011). Nevertheless, it is well known that if the main mineral used by invertebrates for hardening their skeleton is calcium carbonate, numerous other minerals are also found, notably phosphate and silica-based minerals Knoll, 2003;Lowenstam, 1972;Lowenstam and Weiner, 1989;Murdock and Donohue, 2011;Simkiss and Wilbur, 1989).…”