Expression, activation, and processing of the recombinant snake venom metalloproteinase, pro-atrolysin E.
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.
Shimokawa
K
K
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Jia
L G
LG
Wang
X M
XM
Fox
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D003435:Crotalid Venoms / Q000378:metabolism*; D017839:Crotalus; D004792:Enzyme Precursors / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism
D003435:Crotalid Venoms; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C044577:atrolysin A; C095664:atrolysin E