ADAM-9 is an insulin-like growth factor binding protein-5 protease produced and secreted by human osteoblasts
ADAM-9 is an insulin-like growth factor binding protein-5 protease produced and secreted by human osteoblasts
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DOI：
10.1021/bi026458q
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发表时间：
2002-12-24
期刊：
影响因子：
2.9
通讯作者：
Baylink, DJ
中科院分区：
文献类型：
--
作者：
Mohan, S;Thompson, GR;Baylink, DJ
IGF结合蛋白-5 （BP-5）是一种重要的骨形成调节因子。因此，阐明成骨细胞产生的IGF结合蛋白-5 （BP-5）蛋白酶的身份对于我们理解控制BP-5作用的分子途径非常重要。因此，从U2人骨肉瘤细胞的条件培养基中经过不同色谱步骤纯化的BP-5蛋白酶作为单个主条带迁移，在还原条件下与天然PAGE中的蛋白酶活性相结合，在SDS-PAGE中产生多个条带。这些条带的n端测序显示，其中三个条带产生的氨基酸序列与alpha2巨球蛋白（alpha2M）相同。虽然alpha2M是由人成骨细胞（OBs）产生的，但没有发现它是BP-5蛋白酶。由于alpha2M已被证明与ADAM蛋白酶复合物，并且发现ADAM-12可切割BP-3和BP-5，因此我们评估了ADAM家族的一个成员是否为BP-5蛋白酶。基于以下发现：(1)U2细胞CM中纯化的BP-5蛋白酶制剂中含有ADAM-9， (2) ADAM-9在各种OB细胞类型中大量产生和分泌，(3)纯化的ADAM-9能有效地切割BP-5，而不切割其他igfbp或效率较低，(4)纯化的ADAM-9能与alpha2M结合，我们认为ADAM-9是人OB产生的BP-5蛋白酶。
IGF binding protein-5 (BP-5) is an important bone formation regulator. Therefore, elucidation of the identity of IGF binding protein-5 (BP-5) protease produced by osteoblasts is important for our understanding of the molecular pathways that control the action of BP-5. In this regard, BP-5 protease purified by various chromatographic steps from a conditioned medium of U2 human osteosarcoma cells migrated as a single major band, which comigrated with the protease activity in native PAGE and yielded multiple bands in SDS-PAGE under reducing conditions. N-Terminal sequencing of these bands revealed that three of the bands yielded amino acid sequences that were identical to that of alpha2 macroglobulin (alpha2M). Although alpha2M was produced by human osteoblasts (OBs), it was not found to be a BP-5 protease. Because alpha2M had been shown to complex with ADAM proteases and because ADAM-12 was found to cleave BP-3 and BP-5, we evaluated if one of the members of ADAM family was the BP-5 protease. On the basis of the findings that (1) purified preparations of BP-5 protease from U2 cell CM contained ADAM-9, (2) ADAM-9 is produced and secreted in high abundance by various human OB cell types, (3) purified ADAM-9 cleaved BP-5 effectively while it did not cleave other IGFBPs or did so with less potency, and (4) purified ADAM-9 bound to alpha2M, we conclude that ADAM-9 is a BP-5 protease produced by human OBs.