Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein

Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca2 +-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca2 + with high affinity (Kd = 1.46 nM) and favourable Gibbs free energy (∆G = − 12.4 kcal/mol). The stoichiometry for Ca2 + bound to sbCRTAC2 at saturation indicated six Ca2 + ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca2 +. Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25 °C and 95 °C and the fully unfolded state is only induced by chemical denaturing (4 M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell–cell and cell–matrix interactions.

► CRTAC2 is mainly composed by β-sheet structures which favour its hyperthermostability.
► CRTAC2 binds Ca2 + with high affinity probably via integrin-like Ca2 +-binding motifs.
► Protein aggregates of teleost CRTAC2 exist in vivo and in vitro.
► CRTAC2 promotes epithelial cell outgrowth in vitro.