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Volume 270, Number 22, Issue of June 2, pp. 13496-13502, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Ralph Melchior , James P. Quigley , Peter B. Armstrong

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 -macroglobulin (M), which binds active proteases, and the M-protease complex is then cleared from the plasma by a receptor-mediated endocytotic process. M 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 M, and that was precipitated by antibodies directed against Limulus M. 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 (t > 180 min). FITC-labeled, trypsin-reacted Limulus M was cleared rapidly from the plasma of Limulus, whereas FITC-labeled, native Limulus M persisted undiminished in excess of 400 min. The blood cells of Limulus bound FITC-labeled trypsin-reacted Limulus M, 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 M-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 M is the probable agent for protease clearance. This is the first documentation of a protease clearance pathway in invertebrates and represents the first identified physiological function for M in invertebrates.

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