THE PHOSPHATIDYLINOSITOL-GLYCOLIPID ANCHOR ON ALKALINE-PHOSPHATASE FACILITATES MINERALIZATION INITIATION IN-VITRO

Alkaline phosphatase (AP) is required for the proper mineralization of cartilage and bone. The enzyme is localized to the outer surface of c ells through a phosphatidylinositol-glycolipid anchor, which is covale ntly attached to the carboxyl terminus of the protein. In calcifying c artilage, AP-rich matrix vesicles (MVs) are release into the matrix fr om chondrocytes, and apatite formation is initiated within and around these particles. In this paper we examine the role of the AP glycolipi d anchor using an in vitro mineralization assay system. AP was purifie d to homogeneity, and the purified enzyme was used to drive mineral fo rmation in vitro with and without the anchor. Mineral formation was in itiated through phosphate release from beta-glycerol phosphate (beta-G P). The amount of PO4-3 released was similar whether the anchor was pr esent or absent. However, SEM and X-ray microanalysis revealed that th e mineral produced by anchored AP was indistinguishable from that prod uced by MVs and that both of those minerals were more apatite-like tha n mineral formed by soluble AP or through spontaneous precipitation. T aken together, the data suggest that in addition to providing PO4-3 to drive mineralization, AP influences the nature of the mineral formed. Further, AP containing its glycolipid anchor produces mineral compara ble with that formed by tissue-derived MVs. Thus, in the absence of ex tracellular matrix, MV mineralization in vitro can be emulated by glyc olipid-anchor containing AP.