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 cells through a phosphatidylinositol-glycolipid anchor, which is covalently attached to the carboxyl terminus of the protein. In calcifying cartilage, AP-rich matrix vesicles (MVs) are released into the matrix from chondrocytes, and apatite formation is initiated within and around these particles. In this paper we examine the role of the AP glycolipid anchor using an in vitro mineralization assay system. AP was purified to homogeneity, and the purified enzyme was used to drive mineral formation in vitro with and without the anchor. Mineral formation was initiated through phosphate release from beta-glycerol phosphate (beta-GP). The amount of PO4(-3) released was similar whether the anchor was present or absent. However, SEM and X-ray microanalysis revealed that the mineral produced by anchored AP was indistinguishable from that produced by MVs and that both of those minerals were more apatite-like than mineral formed by soluble AP or through spontaneous precipitation. Taken 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 comparable with that formed by tissue-derived MVs. Thus, in the absence of extracellular matrix, MV mineralization in vitro can be emulated by glycolipid-anchor containing AP.
Harrison
G
G
Department of Biochemitry, University of Pennsylvania School of Dental Medicine, Philadelphia, USA.
Shapiro
I M
IM
Golub
E E
EE
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000502:physiology*; D002645:Chickens; D004577:Electron Probe Microanalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005994:Glycerophosphates / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D012015:Reference Standards; D012984:Software; D013977:Tibia / Q000201:enzymology / Q000648:ultrastructure
D005994:Glycerophosphates; D017261:Glycosylphosphatidylinositols; D010710:Phosphates; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; C031463:beta-glycerophosphoric acid