Structural Basis of Calcification Inhibition by α2-HS Glycoprotein/Fetuin-A

Genetic evidence from mutant mice suggests that alpha(2)-HS glycoprotein/fetuin-A (Ahsg) is a systemic inhibitor of precipitation of basic calcium phosphate preventing unwanted calcification. Using electron microscopy and dynamic light scattering, we demonstrate that precipitation inhibition by Ahsg is caused by the transient formation of soluble, colloidal spheres, containing Ahsg, calcium, and phosphate. These "calciprotein particles" of 30-150 nm in diameter are initially amorphous and soluble but turn progressively more crystalline and insoluble in a time- and temperature-dependent fashion. Solubilization in Ahsg-containing calciprotein particles provides a novel conceptual framework to explain how insoluble calcium precipitates may be transported and removed in the bodies of mammals. Mutational analysis showed that the basic calcium phosphate precipitation inhibition activity resides in the amino-terminal cystatin-like domain D1 of Ahsg. A structure-function analysis of wild type and mutant forms of cystatin-like domains from Ahsg, full-length fetuin-B, histidine-rich glycoprotein, and kininogen demonstrated that Ahsg domain D1 is most efficient in inhibiting basic calcium phosphate precipitation. The computer-modeled domain structures suggest that a dense array of acidic residues on an extended beta-sheet of the cystatin-like domain Ahsg-D1 mediates efficient inhibition.