We present data suggesting a function of ␣ 2 -HS glycoproteins/fetuins in serum and in mineralization, namely interference with calcium salt precipitation. Fetuins occur in high serum concentration during fetal life. They accumulate in bones and teeth as a major fraction of noncollagenous bone proteins. The expression pattern in fetal mice confirms that fetuin is predominantly made in the liver and is accumulated in the mineralized matrix of bones. We arrived at a hypothesis on the molecular basis of fetuin function in bones using primary rat calvaria osteoblast cultures and salt precipitation assays. Our results indicate that fetuins inhibit apatite formation both in cell culture and in the test tube. This inhibitory effect is mediated by acidic amino acids clustering in cystatin-like domain D1. Fetuins account for roughly half of the capacity of serum to inhibit salt precipitation. We propose that fetuins inhibit phase separation in serum and modulate apatite formation during mineralization.

We proposed that the ␣ 2 -Heremans Schmid glycoprotein/fetuin family of serum proteins inhibits unwanted mineralization. To test this hypothesis in animals, we cloned the mouse fetuin gene and generated mice lacking fetuin. The gene consists of seven exons and six introns. The cystatin-like domains D1 and D2 of mouse fetuin are encoded by three exons each, whereas a single terminal exon encodes the carboxyl-terminal domain D3. The promoter structure is well conserved between rat and mouse fetuin genes within the regions shown to bind transcription factors in the rat system. Expression studies demonstrated that mice homozygous for the gene deletion lacked fetuin protein and that mice heterozygous for the null mutation produced roughly half the amount of fetuin protein produced by wild-type mice. Fetuin-deficient mice were fertile and showed no gross anatomical abnormalities. However, the serum inhibition of apatite formation was compromised in these mice as well as in heterozygotes. In addition, some homozygous fetuin-deficient female ex-breeders developed ectopic microcalcifications in soft tissues. These results corroborate a role for fetuin in serum calcium homeostasis. The fact that generalized ectopic calcification did not occur in fetuin-deficient mice proves that additional inhibitors of phase separation exist in serum.

alpha 2-HS glycoprotein (alpha 2-HS) is a major protein occurring in human blood and calciferous tissues. Due to extensive sequence identity, alpha 2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native alpha 2-HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N-glycosylations and O-glycosylations, and sulfation of the carbohydrate side chains. Various two-chain forms of alpha 2-HS have been prepared from human plasma, however, the single-chain precursor has not yet been isolated. Here, we have studied the biosynthesis of alpha 2-HS by a human hepatoma cell line, HepG2. We demonstrate that a single-chain form and the two-chain form of alpha 2-HS are secreted by this cell line. The alpha 2-HS forms are further modified by phosphorylation on multiple serine residues. Mapping studies indicate that the connecting peptide region releasable from the heavy chain of alpha 2-HS contains at least one such phosphorylation site. Our results identify proteolytic trimming and/or phosphorylation as modifications possibly regulating the biological effects of alpha 2-HS and the homologous fetuins.

α2‐HS glycoprotein (α2‐HS) is a major protein occuring in human blood and calciferous tissues. Due to extensive sequence identity, α2‐HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native α2‐HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N‐glycosylations and O‐glycosylations, and sulfation of the carbohydrate side chains. Various two‐chain forms of α2‐HS have been prepared from human plasma, however, the single‐chain precursor has not yet been isolated. Here, we have studied the biosynthesis of α2‐HS by a human hepatoma cell line, HepG2. We demonstrate that a single‐chain form and the two‐chain form of α2‐HS are secreted by this cell line. The α2‐HS forms are further modified by phosporylation on multiple serine residues. Mapping studies indicate that the connecting peptide region releasable from the heavy chain of α2‐HS contains at least one such Phosphorylation site. Our results identify proteolytic trimming and/or phosphorylation as modifications possibly regulating the biological effects of α2‐HS and the homologous fetuins.