Expression, Activation, and Processing of the Recombinant Snake Venom Metalloproteinase, Pro-Atrolysin E

Author： Shimokawa K.I. Jia L.G. Wang X.M. Fox J.W.

Publisher： Elsevier

ISSN： 0003-9861

Source： Archives of Biochemistry and Biophysics, Vol.335, Iss.2, 1996-11, pp. : 283-294

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Abstract

The expression in human embryonic kidney (HEK 293) cells of the recombinant zymogen form (pro-) of the Crotalus atrox hemorrhagic metalloproteinase, atrolysin E, is presented. The nascent protein is comprised of pre-, pro-, proteinase-, spacer-, and disintegrin domains. The biochemical characterization of the recombinant zymogen is described along with its activation by C. atrox crude venom and other hemorrhagic toxins. Unlike the zymogen forms of the matrix metalloproteinases, pro-atrolysin E is not activated by the organomercurial, (4-aminophenyl)mercuric acetate. Pro-atrolysin E could be enzymatically activated by C. atrox crude venom, PMSF-inhibited crude venom, atrolysin A, and atrolysin E itself. There is no evidence of autoactivation. Using two polyclonal antibodies directed against the proteinase domain and the disintegrin domain of atrolysin E, the proteolytic processing of the recombinant protein by atrolysin A was followed. The first cleavage of pro-atrolysin E by atrolysin A removes the pro-domain. The second proteolysis step removes the disintegrin domain to produce the proteinase/spacer protein. These studies have identified potential activators of snake venom pro-metalloproteinases in crude venom and suggest a general scheme for the activation and processing of venom pro-metalloproteinases by the endogenous, active metalloproteinases.