Identification of the cleavage sites by a hemorrhagic metalloproteinase in type IV collagen.
Type IV collagen, solubilized from Engelbreth-Holm-Swarm (EHS) tumor basement membranes is digested by a hemorrhagic metalloproteinase, Ht-e, isolated from the crude venom of the Western Diamondback rattlesnake, Crotalus atrox. The major proteolytic products have Mr 141,000, 132,000, 87,000, 71,000, 33,000 and approximately 18,000 as estimated by SDS-gel electrophoresis of pepsinized type IV collagen fragments. Sequence analysis of the digestion products reveal that the Mr 141,000, 71,000 and approximately 18,000 band are derived from the alpha 1(IV) chains and the Mr 132,000, 87,000 and 33,000 bands are derived from the alpha 2(IV) chain. The products are stable over 72-hour incubation periods. The cleavage sites on the alpha 1(IV) and alpha 2(IV) chains are not identical. The alpha 1(IV) chains are cleaved in a pepsin susceptible triplet interruption region of the triple helix at position Ala258-Gln259. The alpha 2(IV) chain is cleaved in the triple helical region near the NC2 domain at the Gly191-Leu192 peptide bond. Isolated hexameric NC1 globular domains of type IV collagen are not digested by Ht-e. The present study demonstrates that the venom hemorrhagic metalloproteinase Ht-e has type IV collagenolytic activity. The triple helix of the type IV collagen molecule is cleaved in a region located immediately carboxyl to the flexible NC2 domain. The degradation by Ht-e of type IV collagen, a major component of basement membranes which forms the scaffold of this extracellular structure, may account in part for the hemorrhagic activity of this toxin.
Baramova
E N
EN
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Shannon
J D
JD
Bjarnason
J B
JB
Fox
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D003094:Collagen / Q000378:metabolism*; D003435:Crotalid Venoms; D008666:Metalloendopeptidases; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight
D003435:Crotalid Venoms; C032814:hemorrhagin components (Crotalid); D003094:Collagen; D008666:Metalloendopeptidases