α2-Macroglobulin-mediated Clearance of Proteases from the Plasma of the American Horseshoe Crab, Limulus polyphemus

Abstract

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.

Localisation of the major reactive lysine residue involved in the selfcrosslinking of proteinase‐activated Limulusα2‐macroglobulin

“…Hence, the formation of specific intramolecular crosslinks in Limulus a2M when reacted with a proteinase is likely to represent an alternative mechanism for stabilising the complex, fulfilling the same functional role as the intermolecular crosslinking of a-macroglobulins in other species [28]. This is supported by recent results showing that injected proteinase or Limulus a2M-proteinase complexes are cleared in 10-30 min in vivo [29] which is considerably faster than the time it takes to run a non-denaturing gel (about 3 h), indicating that the complex is sufficiently stable to be cleared from circulation without leaking the non-covalently trapped proteinase.…”

Effect of dopamine injection on the hemocyte count and prophenoloxidase system of the white shrimp Litopenaeus vannamei

“…Cleavage of the bait region by a proteinase from any mechanistic class (not only SP) leads to a conformational change that traps the proteinase in a cavity formed by the α 2 -M tetramer (in vertebrates) or dimer (in invertebrates). The change in conformation also leads to formation of covalent crosslinks between the thiolester region of α 2 -M and lysine sidechains of the proteinase, resulting in irreversible inhibition of the proteinase, even though its active site is not acted (Starkey et al, 1982;Laskowski and Kato, 1980;Sottrup-Jensen et al, 1986;Melchior et al, 1995;Gollas-Galván et al, 2003). In the present study, α 2 -M activity increased significantly after injection with DA, reached the maximum in 3 h and reached a stable level after 12 h, and there were no significant differences between the control and experimental groups thereafter.…”

Molecular cloning and expression analysis of α2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus

“…The molecule, which has been isolated in all class of vertebrates and several invertebrates, is a broad-spectrum proteinase-binding protein, which leads to elimination of circulating proteinases in the plasma [23]. Therefore, a 2 M is thought to play an important role in innate immunity [9,11].…”

“…A variety of mammalian cells including hepatocytes, fibroblasts, and monocytes/macrophages, bind to the receptor of proteinasereacted a 2 M [22]. In arthropods, granular amebocytes, a type of blood cell, appear to participate in the clearance of the a 2 M-proteinase complex [23].…”