Wu S M, Boyer C M, Pizzo S V
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1997 Aug 15;272(33):20627-35. doi: 10.1074/jbc.272.33.20627.
Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.
受体识别形式的α2-巨球蛋白（α2M*）与两类细胞受体结合，一类是高亲和力位点，约1500个位点/细胞，另一类是低亲和力位点，约60000个位点/细胞。后一类已被鉴定为所谓的低密度脂蛋白受体相关蛋白（LRP）。与LRP不同的受体的连接激活细胞信号通路。有力的间接证据表明，高亲和力结合位点负责α2M诱导的细胞信号传导。使用次氯酸钠（一种由H2O2-髓过氧化物酶-Cl-系统产生的强氧化剂），我们现在证明与高亲和力位点的结合与信号级联的激活直接相关。用200μM次氯酸钠氧化α2M完全消除了其与LRP的结合，而不影响其激活巨噬细胞信号级联的能力。Scatchard分析显示与一类单一的高亲和力位点结合（Kd - 71 +/- 12 pM）。令人惊讶的是，用125μM次氯酸钠氧化天然α2-巨球蛋白（α2M）会导致其受体结合位点暴露于LRP，但该配体无法诱导细胞信号传导。Scatchard分析显示与一类单一的低亲和力位点结合（Kd - 0.7 +/- 0.15 nM）。α2M（RBF）的克隆和表达的羧基末端20 kDa片段能够与LRP和信号受体结合，其氧化后结合Kd没有显著变化，支持了我们早期的发现，即氧化敏感位点主要在RBF之外。试图理解氧化天然α2M中LRP结合位点选择性暴露的机制表明，部分蛋白质解折叠可能是最可能的机制。这些研究提供了强有力的证据，表明高亲和力位点（Kd - 71 pM）是α2M*信号受体。
