Mineralization by Inhibitor Exclusion: THE CALCIFICATION OF COLLAGEN WITH FETUIN

One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism “mineralization by inhibitor exclusion,” the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization.The type I collagen fibril plays several critical roles in bone mineralization. The mineral in bone is located primarily within the fibril (1–6), and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral (7, 8). The collagen fibril therefore provides the aqueous compartment in which mineral grows. We have recently shown that the physical structure of the collagen fibril plays an important additional role in mineralization, that of a gatekeeper allowing molecules smaller than a 6-kDa protein to freely access the water within the fibril while preventing molecules larger than a 40-kDa protein from entering the fibril (9).Molecules too large to enter the collagen fibril can have important effects on mineralization within the fibril. We have suggested that large inhibitors of apatite growth can paradoxically favor mineralization within the fibril by selectively preventing apatite growth in the solution outside of the fibril (9). We have also proposed that large nucleators of apatite formation may generate small crystals outside the collagen fibril and that some of these crystals can subsequently diffuse into the fibril and grow (9). Because the size exclusion characteristics of the fibril allow rapid penetration of molecules the size of a 6-kDa protein, apatite crystals up to 12 unit cells in size should in principle be able to freely access all of the water within the fibril (9).We subsequently tested these hypotheses for the role of large molecules in fibril mineralization by determining the impact of removing fetuin on the serum-driven calcification of collagen fibrils (10). Fetuin is the most abundant serum inhibitor of apatite crystal growth (11, 12), and with a molecular weight of 48 kDa fetuin is too large to penetrate the collagen fibril (9). Fetuin is also termed fetuin-A (to distinguish it from a recently discovered homologue, fetuin-B (13)) and is sometimes called α2-HS glycoprotein in humans. Our working hypothesis was that fetuin is required for the serum-driven calcification of a collagen fibril and that its role is to favor calcification within the collagen fibril by selectively preventing apatite crystal growth in the solution outside the fibril.The results of this study demonstrate that removing fetuin from serum eliminates the ability of serum to induce the calcification of a type I collagen matrix and that adding purified fetuin to fetuin-depleted serum restores this activity (14). This study further shows that a massive mineral precipitate forms during the incubation of fetuin-depleted serum but not during the incubation of serum containing fetuin (14). These observations are consistent with the hypothesis that a large serum nucleator generates apatite crystals in the solution outside of the collagen fibril, some of which penetrate into the aqueous interior of the fibril (14). Because fetuin can trap only those nuclei that it can access, the crystal nuclei that penetrate the fibril grow far more rapidly than those nuclei trapped by fetuin outside of the fibril, and the collagen fibril therefore selectively calcifies.The goal of the present experiments was to further understand the role of fetuin in the calcification of type I collagen fibrils. To accomplish this goal, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentrations and the only macromolecule in the solution is fetuin. This system allowed us to probe the impact of fetuin and only fetuin on the location and extent of collagen calcification. Because fetuin is the subject of this study, it is useful to review briefly its occurrence and calcification-inhibitory activity. Fetuin is a 48-kDa glycoprotein that is synthesized in the liver and is found at high concentrations in mammalian serum (15, 16) and bone (17–22). The serum fetuin concentration in adult mammals ranges from 0.5 to 1.5 mg/ml, whereas the serum fetuin concentration in the fetus and neonate is typically far higher (16). Fetuin is also one of the most abundant noncollagenous proteins found in bone (17–22), with a concentration of about 1 mg fetuin/g bone in rat (21), bovine (17), and human (19, 23) bone. Despite the abundance of fetuin in bone, however, it has not been possible to demonstrate the synthesis of fetuin in calcified tissues, and it is therefore presently thought that the fetuin found in bone arises from hepatic synthesis via serum (20, 22). This view is supported by the observation that fetuin binds strongly to apatite, the mineral phase of bone, and is selectively concentrated from serum onto apatite (18).In vitro studies have demonstrated that fetuin is an important inhibitor of apatite growth and precipitation in serum containing increased levels of calcium and phosphate (12) and that targeted deletion of the fetuin gene reduces the ability of serum to arrest apatite formation by over 70% (11). More recent studies have shown that a fetuin-mineral complex is formed in the course of the fetuin-mediated inhibition of apatite growth and precipitation in serum containing increased calcium and phosphate (24, 25). Purified fetuin also potently inhibits the growth of apatite crystals from supersaturated solutions of calcium phosphate (12, 24). In solutions in which a decline in calcium occurs within minutes because of the spontaneous formation of apatite crystals, the presence of added fetuin sustains elevated calcium levels for at least 24 h (24).