Interaction of hemorrhagic metalloproteinases with human .alpha.2-macroglobulin

Publisher Website
Google Scholar
Abstract
The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human .alpha.2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by .alpha.2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of .alpha.2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with .alpha.2 - macroglobulin rapidly at 22 .degree.C. Rate constants based on intrinsic fluorescence measurements were 0.62 .times. 105 M-1 s-1 for interaction of .alpha.2-macroglobulin with Ht-c and -d and 2.3 .times. 105 M-1 s-1 for the interaction of .alpha.2-macroglobulin with Ht-e. Ht-a interacted with .alpha.2-macroglobulin very slowly at 22 .degree.C. Increasing the temperature to 37 .degree.C and prolonging the time of interaction with .alpha.2-macroglobulin resulted in the formation of Mr 90 000 fragments and high molecular weight complexes (Mr > 180 000), in which Ht-a is covalently bound to the carboxy-terminal fragment of .alpha.2-M. The identification of the sites of specific proteolysis of .alpha.2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves .alpha.2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified. Ht-e cleaves .alpha.2-macroglobulin at two sites which are different from those observed with Ht-a, -c, and -d. With Ht-e the primary cleavage site is the Val689-Met690 peptide bond and the secondary site at Gly693-His694. Of the four toxins investigated, Ht-a is the most potent hemorrhagic toxin in vivo. We propose that reaction of Ht-a with the primary plasma proteinase inhibitor .alpha.2-M may partially explain the high hemorrhagic activity of this toxin.