Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization

Abstract

The larval spicule matrix protein SM50 is the most abundant occluded matrix protein present in the mineralized larval sea urchin spicule. Recent evidence implicates SM50 in the stabilization of amorphous calcium carbonate (ACC). Here, we investigate the molecular interactions of SM50 and CaCO3 by investigating the function of three major domains of SM50 as small ubiquitin-like modifier (SUMO) fusion proteins - a C-type lectin domain (CTL), a glycine rich region (GRR) and a proline rich region (PRR). Under various mineralization conditions, we find that SUMO-CTL is monomeric and influences CaCO3 mineralization, SUMO-GRR aggregates into large protein superstructures and SUMO-PRR modifies the early CaCO3 mineralization stages as well as growth. The combination of these mineralization and self-assembly properties of the major domains synergistically enable the full-length SM50 to fulfill functions of constructing the organic spicule matrix as well as performing necessary mineralization activities such as Ca(2+) ion recruitment and organization to allow for proper growth and development of the mineralized larval sea urchin spicule.

Mineralization and non-ideality: on nature’s foundry

“…Such biomolecules can also modulate the stability of mineral precursors as well as regulate nucleation and phase transitions reflecting the multiple roles of (bio)molecules as additives in mineralization processes (Verch et al 2011;Rao et al 2014). Here, multiple properties of biomolecular additives such as charge, amino acid/glycan/lipid composition, covalent modifications, conformations, and self-assembly propensity as well as intermolecular synergy play mechanistic roles (Arias and Fernández 2008;Bentov et al 2010;Chang et al 2016a, b;Rao et al 2013Rao et al , 2015.…”

“…Proteinaceous and saccharide additives can also tune the stability of early precursors and favor the formation of a particular phase, i.e. mediate poly(a)morph selection (Cartwright et al 2012;Chang et al 2016a;Gebauer et al 2009;Rao et al 2013Rao et al , 2014. Fig.…”

“…For instance, lysozyme (monomer: 14.3 kDa) and ovalbumin (monomer: 45 kDa) have distinct molar mass, surface area and shapes (Table S1), as well as exhibit distinct aggregation propensities in dependence of ionic and pH conditions (Ianeselli et al 2010). For certain proteins, Ca 2+ ions can modulate self-association states via direct interactions (Shearwin and Winzor 1988;Rao et al 2013;Seto et al 2014), suggesting ion-dependent macromolecular conformation bias during mineralization. Further due to distinct density fluctuations and conformations of crowding agents in topologically-frustrated void spaces, crowding induced by different biomacromolecules can have dissimilar consequences (Samiotakis et al 2009).…”

“…Further, macromolecules regulating biomineralization appear to be biochemically tuned in being conformationally responsive to changes in ionic conditions. For instance, distinct ionic conditions lead to β-turn or β-sheet conformations in the lowcomplexity disordered proteins involved in sea urchin skeletogenesis (Xu and Evans 1999;Rao et al 2013). Interestingly, sequentially similar proteins derived from gluten extracts also exhibit distinct homo-and hetero-association behavior tuned by solution ionic strength (Winzor 1966).…”

“…AUC is a powerful method for the quantitative analysis of molecular size, shape and density, and is useful to understand biochemical processes in complex solutions such as crowded but also interacting media (Cölfen et al 1996Wilson et al 1997). The recombinant fusion protein is based on the Nterminal C-type lectin domain of a key biomineralization protein (SM50) involved in sea urchin skeletogenesis (Rao et al 2013). Sedimentation velocity profiles are measured in the absence and presence of PEG-induced crowding (Fig.…”