Shi Z, Xu W, Loechel F, Wewer U M, Murphy L J
Department of Physiology & Internal Medicine, University of Manitoba, Winnipeg R3E 0W3, Canada.
J Biol Chem. 2000 Jun 16;275(24):18574-80. doi: 10.1074/jbc.M002172200.
Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability. IGFBP-3 is extensively degraded in serum during pregnancy; however, as yet the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins that is highly expressed in placental tissue, was identified as interacting with IGFBP-3. This interaction involved the cysteine-rich domain of ADAM 12. Unlike other members of this family of disintegrin metalloproteases that are membrane proteins, ADAM 12 exists as an alternatively spliced soluble secreted protein. To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells. Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed by Western blotting with anti-IGFBP-3 or anti-ADAM 12. Although minimal proteolysis of IGFBP-3 was observed in conditioned medium from control cells, this was increased approximately 4-fold in conditioned medium from ADAM 12-S-transfected cells. Recombinant ADAM 12-S partially purified from conditioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3. The degradation pattern was similar to that seen with pregnancy serum, and the presence of ADAM 12-S in serum during pregnancy was confirmed. The data suggest that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to the IGFBP-3 protease activity present in pregnancy serum.
胰岛素样生长因子结合蛋白（IGFBP）-3以高亲和力结合胰岛素样生长因子并调节其作用。IGFBP-3的蛋白水解切割可能调节胰岛素样生长因子的生物利用度。在孕期，IGFBP-3在血清中被广泛降解；然而，迄今为止，尚未鉴定出孕期特异性蛋白酶。我们利用酵母双杂交试验和人胎盘cDNA文库来研究与IGFBP-3相互作用的蛋白。解整合素金属蛋白酶-12（ADAM 12），一种在胎盘组织中高表达的金属蛋白酶解整合素家族成员，被鉴定为与IGFBP-3相互作用。这种相互作用涉及ADAM 12富含半胱氨酸的结构域。与该解整合素金属蛋白酶家族的其他成员（膜蛋白）不同，ADAM 12以一种可变剪接的可溶性分泌蛋白形式存在。为了验证ADAM 12与IGFBP-3之间的相互作用，将含有ADAM 12-S cDNA的表达构建体转染到COS-1细胞中。当用抗ADAM 12或抗IGFBP-3抗体进行免疫沉淀，然后用抗IGFBP-3或抗ADAM 12进行蛋白质印迹分析条件培养基时，观察到共沉淀现象。虽然在对照细胞的条件培养基中观察到IGFBP-3的蛋白水解作用极小，但在ADAM 12-S转染细胞的条件培养基中，这种作用增加了约4倍。从肝素-琼脂糖柱上的条件培养基中部分纯化的重组ADAM 12-S也能使IGFBP-3发生蛋白水解。其降解模式与孕期血清中的相似，并且证实了孕期血清中存在ADAM 12-S。数据表明ADAM 12-S具有IGFBP-3蛋白酶活性，并且它可能对孕期血清中存在的IGFBP-3蛋白酶活性有贡献。
