Synthesis and characterization of insulin-like growth factor-binding protein (IGFBP)-7 - Recombinant human mac25 protein specifically binds IGF-I and II
Synthesis and characterization of insulin-like growth factor-binding protein (IGFBP)-7 - Recombinant human mac25 protein specifically binds IGF-I and II
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DOI：
10.1074/jbc.271.48.30322
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发表时间：
1996-11-29
影响因子：
4.8
通讯作者：
Rosenfeld, RG
中科院分区：
文献类型：
--
作者：
Oh, YM;Nagalla, SR;Rosenfeld, RG
mac25 cDNA 最初是从软脑膜细胞克隆的，随后通过差异展示重新分离为在衰老的人乳腺上皮细胞中优先表达的序列。推导的人mac25前肽的氨基酸序列与人胰岛素样生长因子结合蛋白（IGFBP）有20-25％的同一性，表明mac25可能是IGFBP家族的另一个成员。在本研究中，我们在杆状病毒表达系统中生成了重组人mac25（rh-mac25），并评估了其对IGF的亲和力，并评估了mac25基因在人中的表达模式。组织。 I-125-IGF-I 和 I-125-IGF-II 与 rh-mac25 的结合通过非变性聚丙烯酰胺凝胶电泳后的蛋白质印迹以及通过与少至 2 nM rh-mac25 的亲和交联来证明。 rh-mac25 与 I-125-IGF 结合的特异性通过 rh-mac25 与未标记的 IGF 竞争结合来证明，但与 [QAYLL]IGF-II 类似物不竞争，后者对 IGFBP 的亲和力低 100 倍。与IGFBP-3相比，rh-mac25对IGF-I的亲和力至少低5-6倍，对IGF-II的亲和力至少低20-25倍。 mac25 mRNA在多种正常人体组织中均可检测到，但在乳腺癌、前列腺癌、结肠癌和肺癌细胞系中表达降低。综上所述，mac25特异性结合IGF，构成IGFBP家族的新成员IGFBP-7。它在正常组织中的广泛分布和在几种癌细胞中的较低表达表明 IGFBP-7 可能充当生长抑制因子以及 IGF 结合蛋白。
The mac25 cDNA was originally cloned from leptomeningial cells and subsequently reisolated through differential display as a sequence preferentially expressed in senescent human mammary epithelial cells. The deduced amino acid sequence of the human mac25 propeptide shares a 20-25% identity to human insulin-like growth factor-binding proteins (IGFBPs), suggesting that mac25 could be another member of the IGFBP family.In the present study, we have generated recombinant human mac25 (rh-mac25) in a baculovirus expression system and assessed its affinity for IGFs and have evaluated the pattern of expression of the mac25 gene in human tissues. Binding of I-125-IGF-I and I-125-IGF-II to rh-mac25 was demonstrated by Western ligand blotting after nondenaturing polyacrylamide gel electrophoresis and by affinity cross-linking with as little as 2 nM rh-mac25. Specificity of rh-mac25 binding to I-125-IGFs was demonstrated by competition for rh-mac25 binding with unlabeled IGFs, but not with [QAYLL]IGF-II analog, which has 100-fold less affinity for IGFBPs. In comparison with IGFBP-3, rh-mac25 has at least a 5-6-fold lower affinity for IGF-I and 20-25-fold lower affinity for IGF-II. mac25 mRNA was detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines,In conclusion, mac25 specifically binds IGFs and constitutes a new member of the IGFBP family, IGFBP-7. Its wider distribution in normal tissue and lower expression in several cancer cells indicate that IGFBP-7 may function as a growth-suppressing factor, as well as an IGF-binding protein.