Biochemical Characterization of the Serum Fetuin-Mineral Complex

Abstract

The present study was carried out to characterize the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein (MGP) that was initially discovered in serum of rats treated with etidronate and appears to play a critical role in inhibiting calcification in vivo. Fetuin purified from the FMC contains 3.3 mol of protein-bound phosphate. There is 1.3 mg of FMC/ml of serum 6 h after etidronate injection, and the FMC is 46% fetuin and 53% mineral by mass. Formation of the FMC in the first 6 h after etidronate injection does not increase serum fetuin despite the fact that 50% of serum fetuin is associated with the FMC, and clearance of the FMC in the 9 -24-h interval lowers total serum fetuin by 50%. These observations suggest that the fetuin component of the FMC is derived from fetuin initially in serum and that clearance of the FMC removes the associated fetuin from circulation. One additional protein was consistently present in all preparations of the FMC, spp24 (secreted phosphoprotein 24). This 24-kDa protein is similar in domain structure to fetuin and, like fetuin and MGP, contains several residues of phosphoserine and accumulates in bone. Exogenous spp24 associated strongly with the FMC when added to serum containing it. These observations suggest that spp24 may, like fetuin and MGP, play a role in inhibiting calcification.The present experiments are a continuation of our investigations into the mechanisms by which proteins interact specifically with mineral in vivo to prevent the calcification of arteries and other soft tissues. In a previous study (1), we described the discovery of a complex of calcium, phosphate, fetuin, 1 and matrix Gla protein in the serum of rats treated with the bone active bisphosphonate etidronate and showed that the appearance of this complex in serum correlates with the inhibition of bone mineralization by etidronate. The fetuin-mineral complex reaches maximal levels 6 -9 h after etidronate injection and causes a 4-fold increase in total serum calcium without causing any increase in ionic calcium levels.The proteins associated with the fetuin-mineral complex, fetuin and matrix Gla protein (MGP), have both been shown to function as potent inhibitors of calcification in vitro. Fetuin is a 59-kDa protein that consists of two N-terminal cystatin domains and a smaller C-terminal domain. Fetuin is synthesized by the liver and secreted into blood, where it is found at a concentration of ϳ1 mg/ml in the rat (2). Previous studies have demonstrated that fetuin is the major calcification inhibitor found in serum (3, 4). Fetuin is also one of the most abundant noncollagenous proteins found in mammalian bone (5-10), with a concentration of ϳ1 mg of fetuin/g of bone in rat (9). MGP is a small, 10-kDa vitamin K-dependent protein (11) that is secreted by a wide variety of cell types, including vascular smooth muscle cells, and is found in bone at a concentration of 0.2 mg/g in the rat (12). Genetic and biochemical studies ...

The serum protein α2–Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification

“…The underlying mechanism involved a direct interaction with the mineral phase and the prevention of large crystal formation. This was confirmed in vivo by the discovery of Ahsg as the major protein of a high-molecular-weight complex also containing calcium, phosphate, and matrix GLA protein in serum of etidronate-treated rats (41)(42)(43)(44).…”

Nanoparticle-Based Test Measures Overall Propensity for Calcification in Serum

“…[19][20][21] In analogy, fetuin-mineral complexes were found in bisphosphonate-treated hypercalcemic rats. 22 We assume that primary CPPs are the predominant form that can be isolated from freshly drawn blood 19 and that their occurrence reflects imminent or active ongoing pathologic calcification. Reverse logic applies to our serum test: high primary and/or secondary CPP and thus, a high refractive signal and late transition times indicate a high residual capacity of the serum to form CPPs and thus, an intact humoral defense against calcification.…”

Serum Osteoprotegerin as a Screening Tool for Coronary Artery Calcification Score in Diabetic Pre-Dialysis Patients

“…Fetuin-A accelerates the solubility of minerals by forming a fetuin mineral complex (FMC) containing calcium and phosphate (27,34), to participate in the inhibition of ectopic calcification. Measuring FMC instead of fetuin-A might allow us to detect a significant association with CACS.…”