Fowlkes J L, Enghild J J, Suzuki K, Nagase H
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1994 Oct 14;269(41):25742-6.
Insulin-like growth factor binding protein-3 (IG-FBP-3) is degraded by a Zn(2+)-dependent protease(s) produced by human dermal fibroblasts in vitro (Fowlkes, J. (1994) Endocrine J. 2, 63-68). Initial studies using IG-FBP-3-substrate zymography identified several IGFBP-3-degrading proteases with M(r) 52,000-72,000, which were inhibitable by EDTA and were shifted to lower M(r) species after treatment of conditioned medium with an organomercurial, suggesting that they might represent one or more of the matrix metalloproteinases (MMPs). Immunoblotting of conditioned medium demonstrated the presence of proMMP-1 (52 and 55 kDa), proMMP-3 (58 and 60 kDa), and proMMP-2 (72 kDa) whose molecular masses corresponded identically to those of the IGFBP-3-degrading proteases. Degradation of recombinant human (rh) IGFBP-3 by conditioned media was blocked (> 80% inhibition) by tissue inhibitor of metallo-proteinases-1, a specific inhibitor of all MMPs, while removal of MMPs -1, -2, and -3 from conditioned medium by sequential immunoaffinity and gelatin-Sepharose chromatography resulted in the complete loss of IGFBP-3-degrading proteinase activity. Furthermore, human MMP-1, MMP-3, and to a lesser extent MMP-2 degraded rhIGFBP-3 in vitro. Sequence analysis of rhIGFBP-3 cleavage sites produced by MMP-1, -2, or -3 demonstrated that each cleaved within the mid-region of the binding protein, a domain with little or no homology with the other five cloned IGFBPs. These studies suggest that MMPs, beyond their previously described functions as extracellular degrading enzymes, may also exert effects on cellular growth and proliferation via degradation of IGFBP-3, thus enhancing IGF bioavailability.
胰岛素样生长因子结合蛋白3（IGFBP - 3）在体外可被人皮肤成纤维细胞产生的一种锌离子依赖性蛋白酶降解（Fowlkes，J.（1994年），《内分泌杂志》2，63 - 68页）。最初使用IGFBP - 3底物酶谱法的研究鉴定出了几种分子量在52,000 - 72,000的IGFBP - 3降解蛋白酶，它们可被EDTA抑制，在用有机汞处理条件培养基后分子量会向较低分子量形式转变，这表明它们可能代表一种或多种基质金属蛋白酶（MMPs）。对条件培养基进行免疫印迹分析显示存在前MMP - 1（52和55 kDa）、前MMP - 3（58和60 kDa）和前MMP - 2（72 kDa），其分子量与IGFBP - 3降解蛋白酶的分子量完全一致。金属蛋白酶组织抑制剂 - 1（一种所有MMPs的特异性抑制剂）可阻断条件培养基对重组人（rh）IGFBP - 3的降解（抑制率> 80%），而通过连续免疫亲和和明胶 - 琼脂糖层析从条件培养基中去除MMPs - 1、 - 2和 - 3后，IGFBP - 3降解蛋白酶活性完全丧失。此外，人MMP - 1、MMP - 3以及程度较轻的MMP - 2在体外可降解rhIGFBP - 3。对由MMP - 1、 - 2或 - 3产生的rhIGFBP - 3切割位点进行序列分析表明，每种酶都在结合蛋白的中间区域进行切割，该区域与其他五个克隆的IGFBPs几乎没有或没有同源性。这些研究表明，MMPs除了其先前描述的作为细胞外降解酶的功能外，还可能通过降解IGFBP - 3对细胞生长和增殖产生影响，从而提高IGF的生物利用度。
