Lalou C, Lassarre C, Binoux M
Unité de Recherches sur la Régulation de la Croissance, Institut National de la Santé et de la Recherche Médicale Unit 142, Hôpital Saint Antoine, Paris, France.
Endocrinology. 1996 Aug;137(8):3206-12. doi: 10.1210/endo.137.8.8754741.
Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like growth factor (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3. Two major fragments of 22/25 kilodaltons (kDa) and one of 16 kDa were identified by Western immunoblotting and isolated by reverse-phase chromatography. The 22/25-kDa fragments correspond to the major approximately 30-kDa glycosylated fragment of IGFBP-3 in serum and the 16-kDa fragment, to one of the same size, that is nonglycosylated. Western ligand blot analysis, affinity cross-linking, and competitive binding experiments using radiolabeled IGF and unlabeled IGF-I or -II showed that in the high performance liquid chromatography eluate containing the 16-kDa fragment, all affinity for IGFs had been lost, whereas the affinity of the 22/25-kDa fragments was considerably reduced. Scatchard analysis of the data indicated a 20-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGFBP-3. In a chick embryo fibroblast assay in which DNA synthesis was stimulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 was found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBPs. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimulation was achieved and no inhibition of insulin stimulation. Unexpectedly, with the fraction containing the 16-kDa fragment (despite the total lack of affinity for IGF-I), IGF-I-induced stimulation was inhibited to nearly the same extent as with intact IGFBP-3. In addition, insulin-induced stimulation was inhibited with similar potency. IGFBP-3 proteolysis therefore generates two types of fragment with different activities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.
胰岛素样生长因子结合蛋白-3（IGFBP-3）的有限蛋白水解日益被认为是调节胰岛素样生长因子（IGF）生物利用度的关键机制，这一过程在血液循环和细胞水平上均有发生。与多种细胞类型接触时产生的纤溶酶会引发蛋白水解切割，这些切割与（尚未明确的）IGFBP-3蛋白酶在体内诱导产生的切割相似。我们确定了实验条件，以实现纤溶酶诱导的重组人非糖基化IGFBP-3的有限蛋白水解。通过蛋白质免疫印迹法鉴定出两个主要的22/25千道尔顿（kDa）片段和一个16 kDa片段，并通过反相色谱法进行分离。22/25-kDa片段对应于血清中IGFBP-3主要的约30-kDa糖基化片段，而16-kDa片段与同样大小的非糖基化片段相对应。使用放射性标记的IGF以及未标记的IGF-I或-II进行的蛋白质印迹配体分析、亲和交联和竞争性结合实验表明，在含有16-kDa片段产物的高效液相色谱洗脱液中，对IGF的所有亲和力均已丧失，而22/25-kDa片段的亲和力则大幅降低。对数据的Scatchard分析表明，与重组人IGFBP-3相比，对IGF-II的亲和力丧失了20倍，对IGF-I的亲和力丧失了50倍。在鸡胚成纤维细胞实验中，IGF-I和胰岛素（浓度为100倍，因此会与1型IGF受体发生相互作用）均可刺激DNA合成，结果发现IGFBP-3几乎完全抑制IGF-I诱导的刺激。它对胰岛素诱导的刺激没有影响，因为胰岛素对IGFBPs没有亲和力。对于22/25-kDa片段，仅实现了对IGF-I刺激的50%抑制，且对胰岛素刺激没有抑制作用。出乎意料的是，对于含有16-kDa片段的组分（尽管对IGF-I完全缺乏亲和力），IGF-I诱导的刺激受到的抑制程度与完整的IGFBP-3几乎相同。此外，胰岛素诱导的刺激也受到类似程度的抑制。因此，IGFBP-3蛋白水解产生了两种具有不同活性的片段。一种对IGF-I具有弱亲和力，只是IGF作用的弱拮抗剂。另一种对IGF缺乏亲和力，但仍能抑制IGF刺激的细胞增殖，因此其作用机制独立于IGF。
