Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC).
Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.
Kelm
R J
RJ
Dept. of Biochemistry, University of Vermont, Burlington 05405.
Hair
G A
GA
Mann
K G
KG
Grant
B W
BW
D000328:Adult; D001792:Blood Platelets / Q000502:physiology*; D001853:Bone Marrow / Q000502:physiology; D001854:Bone Marrow Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000502:physiology; D006026:Glycoside Hydrolases; D006801:Humans; D008533:Megakaryocytes / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010006:Osteoblasts / Q000502:physiology*; D015676:Osteonectin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012516:Osteosarcoma; D014471:Umbilical Veins
D015676:Osteonectin; D006026:Glycoside Hydrolases