Kelm R J, Hair G A, Mann K G, Grant B W
Dept. of Biochemistry, University of Vermont, Burlington 05405.
Blood. 1992 Dec 15;80(12):3112-9.
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.
骨连接素是一种黏附性、细胞及细胞外基质结合糖蛋白，主要存在于体内的骨基质和血小板中。从这两种来源分离出的骨连接素在其组成的N-连接寡糖的复杂性方面存在差异。在本研究中，对体外由成骨细胞（骨母细胞）和血小板生成细胞（巨核细胞）合成的骨连接素进行了分析，以确定所产生的蛋白质在糖基化方面是否分别与从骨和血小板中分离出的蛋白质类似。对成骨样细胞系SaOS-2和MG-63产生的骨连接素进行免疫印迹分析表明，该分子的分泌形式和细胞内形式在结构上是不同的。在血清饥饿条件下，对SaOS-2和MG-63细胞分泌的骨连接素进行内切糖苷酶处理和免疫印迹分析，结果表明该分子具有复杂型寡糖，这与在骨基质来源的骨连接素上发现的高甘露糖部分不同。对SaOS-2细胞和人巨核细胞进行生物合成标记表明，这两种细胞类型都能从头合成骨连接素。对从代谢标记的SaOS-2细胞和巨核细胞裂解物中分离出的[35S]-骨连接素进行电泳和糖苷酶敏感性分析表明，这两种细胞类型合成的骨连接素分子在寡糖结构上与分离出的骨和血小板蛋白相同。这些数据表明，骨连接素分子的细胞内形式在SaOS-2细胞和巨核细胞中的糖基化方式不同，但体内从血小板分泌以及体外从骨母细胞分泌的细胞外形式的特征是存在复杂型N-连接寡糖。
