Melchior R, Quigley J P, Armstrong P B
Department of Molecular and Cellular Biology, University of California, Davis 95616-8755, USA.
J Biol Chem. 1995 Jun 2;270(22):13496-502. doi: 10.1074/jbc.270.22.13496.
Because proteases free in the body are damaging to the tissues, animals have evolved various agents for their inactivation and clearance. Mammals, for instance, have a diverse array of active site protease inhibitors in the plasma. In addition, mammals have alpha 2-macroglobulin (alpha 2M), which binds active proteases, and the alpha 2M-protease complex is then cleared from the plasma by a receptor-mediated endocytotic process. alpha 2M is also present in the plasma of many invertebrates, and in the American horseshoe crab, Limulus polyphemus, it is the only protease inhibitor in the plasma. To search for a clearance process for proteases in Limulus, fluorescein isothiocyanate (FITC)-labeled proteins were injected into the blood, and the fluorescence in the plasma and associated with the blood cells was determined. FITC-labeled trypsin was cleared with an initial mixing period (0-10 min) and a rapid clearance period (10-30 min), followed by the reappearance of FITC in the plasma (45-90 min). Before and during the clearance process, the labeled trypsin was associated with a complex having a molecular mass identical to that of Limulus alpha 2M, and that was precipitated by antibodies directed against Limulus alpha 2M. The fluoresceinated material that reappeared in the plasma after 45 min was of low molecular mass (< 10 kDa) and thus appears to have experienced degradation. The clearance of trypsin requires its protease activity, since phenylmethylsulfonyl fluoride-inactivated, FITC-labeled trypsin was cleared only very slowly if at all (t1/2 > 180 min). FITC-labeled, trypsin-reacted Limulus alpha 2M was cleared rapidly from the plasma of Limulus, whereas FITC-labeled, native Limulus alpha 2M persisted undiminished in excess of 400 min. The blood cells of Limulus bound FITC-labeled trypsin-reacted Limulus alpha 2M, and the peak of recovery from the blood cells coincided with the minimum concentration of FITC-labeled protein in the plasma, suggesting that the blood cells participate in the clearance of alpha 2M-protease complex from the plasma. Thus, we have demonstrated the existence of a clearance pathway in Limulus that operates selectively on enzymatically active proteases and have shown that Limulus alpha 2M is the probable agent for protease clearance. This is the first documentation of a protease clearance pathway in invertebrates and represents the first identified physicological function for alpha 2M in invertebrates.
由于体内游离的蛋白酶会对组织造成损害，动物进化出了各种使其失活和清除的因子。例如，哺乳动物血浆中有多种活性位点蛋白酶抑制剂。此外，哺乳动物还有α2-巨球蛋白（α2M），它能结合活性蛋白酶，然后α2M-蛋白酶复合物通过受体介导的内吞过程从血浆中清除。α2M也存在于许多无脊椎动物的血浆中，在美洲鲎（Limulus polyphemus）中，它是血浆中唯一的蛋白酶抑制剂。为了寻找鲎体内蛋白酶的清除过程，将异硫氰酸荧光素（FITC）标记的蛋白质注入血液，并测定血浆中和与血细胞相关的荧光。FITC标记的胰蛋白酶在初始混合期（0 - 10分钟）和快速清除期（10 - 30分钟）被清除，随后FITC在血浆中重新出现（45 - 90分钟）。在清除过程之前和期间，标记的胰蛋白酶与一种分子量与鲎α2M相同的复合物相关联，并且该复合物可被针对鲎α2M的抗体沉淀。45分钟后在血浆中重新出现的荧光物质分子量较低（< 10 kDa），因此似乎经历了降解。胰蛋白酶的清除需要其蛋白酶活性，因为苯甲基磺酰氟失活的、FITC标记的胰蛋白酶即使有清除也非常缓慢（t1/2 > 180分钟）。FITC标记的、与胰蛋白酶反应的鲎α2M从鲎的血浆中迅速清除，而FITC标记的天然鲎α2M在超过400分钟的时间内持续存在且未减少。鲎的血细胞结合FITC标记的、与胰蛋白酶反应的鲎α2M，并且从血细胞中回收的峰值与血浆中FITC标记蛋白质的最低浓度一致，这表明血细胞参与了α2M-蛋白酶复合物从血浆中的清除。因此，我们证明了鲎体内存在一种对酶活性蛋白酶具有选择性作用的清除途径，并表明鲎α2M可能是蛋白酶清除的介质。这是无脊椎动物中蛋白酶清除途径的首次记录，也是α2M在无脊椎动物中首次确定的生理功能。
