Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin
Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin
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DOI：
10.1042/0264-6021:3580359
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发表时间：
2001-09-01
影响因子：
4.1
通讯作者：
Oxvig, C
中科院分区：
文献类型：
--
作者：
Boldt, HB;Overgaard, MT;Oxvig, C
胰岛素样生长因子 (IGF)-I 和 -II 的生物利用度由六种 IGF 结合蛋白 (IGFBP 1-6) 控制。结合的 IGF 没有活性，但结合蛋白的蛋白水解会导致 IGF 释放。最近发现妊娠相关血浆蛋白 A (PA-PP-A) 以 IGF 依赖性方式裂解 IGFBP-4。为了通过实验支持 PAPP-A 属于金属蛋白酶 metzincin 超家族（均含有延长的锌结合基序 HEXXHXXGXXH（PAPP-A 中的 His-482-His-492））的假设，我们在哺乳动物细胞中表达了 PAPP-A 的突变体。用 Ala 取代 Glu-483 会导致活性完全丧失，将该基序定义为 PAPP-A 活性位点的一部分。有趣的是，Glu-483 被 Gln 取代的突变体显示出残留活性。已知的metzincin结构含有所谓的Met-turn，其严格保守的Met残基被认为与活性位点的残基直接相互作用。通过进一步诱变，我们提供了实验证据，证明 PAPP-A 的 Met-556（来自锌结合基序的 63 个残基）位于 PAPP-A 的 Met 转角中。我们的假设也得到了二级结构预测以及 55 个残基缺失突变体 (d[S498-Y552]) 表达和保留抗原性的能力的支持。然而，由于 PAPP-A 在定义各个已建立的 metzincin 家族的特征方面有所不同，因此我们建议 PAPP-A 属于一个单独的家族。我们还发现 PAPP-A 可以进行自动切割，并且自动切割后的 PAPP-A 没有活性。发现的 PAPP-A 和变体切割位点周围的序列中缺乏统一元件，表明底物特异性存在空间调控。
The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PA-PP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.