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

Abstract

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.amorphous calcium carbonate (ACC) ͉ biomineralization ͉ RNAi ͉ Crustacea

Solubility and bioavailability of stabilized amorphous calcium carbonate

“…(18) Based on our findings, phosphoproteins in this remarkable organism were found to be capable of stabilizing one of the least stable salts in nature, ACC. (17) The unique physicochemical properties of ACC enable the crayfish to store large quantities of calcium in transient storage sites and, when needed, to rapidly and efficiently reabsorb calcium through the intestinal epithelia. These observations led us to the hypothesis that ACC also may be absorbed effectively in higher organisms.…”

“…In an attempt to further increase the similarity of synthetically produced ACC to the ACC produced by the crayfish, embedded in a chitin matrix and serving as our biologic inspiration, (17,18) chitosan, the commonly used deacetylated derivative of chitin, was added during the synthesis of ACC-C. Chitosan is also suggested to increase intestinal absorption of macromolecules such as octreotide, insulin, and low-molecular-weight heparin (LMWH). (45)(46)(47) It is proposed that polycations such as chitosan increase molecule absorption by enhancing permeability through tight junctions in the intestine.…”

“…The role of the chitin and the proteins in ACC stabilization, and possibly also in calcium absorption, has been investigated only recently. (16,17) When needed, the thermodynamic instability of ACC can be exploited to dissolve calcium and allow for fast transport of this ion across the intestinal epithelium and into the bloodstream. (18,19) The bioavailability of calcium in both humans and rats has been suggested to be governed by factors such as dose quantity, the nature of the chelating molecule, solubility of the salt, and the hormonal state of the receiving subject.…”

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).…”

“…Phosphorylations can constitute such important posttranslational modifications interacting with calcification processes (George and Veiss, 2008;Hecker et al, 2003;Inoue et al, 2001Inoue et al, , 2004Shechter et al, 2008b).…”