Human neutrophil collagenase cleaves x 1 - antitrypsin

Inactivation of the plasma serine-proteinase inhibitor a1-antitrypsin (a1-AT) by neutrophil metalloproteinases has been reported [Vissers, George, Bathurst, Brennan & Winterbourn (1987) Fed. Proc. Fed. Am. Soc. Exp. Biol. 46, 1390a; (1988) J. Clin. Invest. 82, 706-711; Desrochers & Weiss (1988) J. Clin: Invest. 81, 1646-1650]. To identify the enzyme responsible, supernatant from neutrophils stimulated with phorbol 12-myristate 13-acetate was subjected to preparative SDS/PAGE, both with and without activation of latent metalloproteinases with HgCl2. The lanes were subsequently sliced into pieces, the slices incubated with equimolar amounts of type I collagen and a1-AT in the presence of HgCl2, and the reaction products separated by SDS/PAGE. With the latent supernatant, the characteristic collagencleavage products and cleaved a1-AT were present in the same slices, corresponding to an Mr of 80000-85000. On treatment with HgCl2 both degradative activities underwent the same molecular-mass shift to a position corresponding to Mr 60000-65000. Western blots of neutrophil supernatants, using a polyclonal antibody to purified collagenase, showed Mr values of 83 000 for the latent enzyme and 63 000 for the HgCl2-activated enzyme. Neutrophil collagenase was purified to homogeneity and shown also to exist in a second latent form with Mr 70000. When activated to the Mr-63000 form by HgCI2 and incubated with equimolar amounts of collagen and a1-AT, collagenase cleaved a,-AT at almost twice the rate at which collagen was cleaved. a1-AT cleavage was inhibited by 1,10-phenanthroline and by high concentrations of collagen. That the purified collagenase did not contain a contaminant proteinase such as stromelysin was indicated by inability of the preparation to cleave casein. Taken together these results lead us to conclude that neutrophil collagenase is capable of degrading al-AT. Neutrophil gelatinase also cleaved a,-AT, but cleavage was slow when compared with its activity against gelatin.