Identification of the Cleavage Sites by a Hemorrhagic Metalloproteinase in Type IV Collagen

Abstract

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 141000, 132000, 87000, 71 000, 33000 and ~ 18000 as estimated by SDS-gel electrophoresis of pepsinized type IV collagen fragments.Sequence analysis of the digestion products reveal that the Mr 141000, 71 000 and ~ 18 000 bands are derived from tha αl (IV) chains and the Mr 132000, 87000 and 33000 bands are derived from the α2(IV) chain.The products are stable over 72-hour incubation periods.The cleavage sites on the αl(IV) and α2(IV) chains are not identical.The αl (IV) chains are cleaved in a pepsin susceptible triplet interruption region of the triple helix at position Ala258-Gln259.The α2(IV) chain is cleaved in the triple helical region near the NC2 domain at the G1Y191Leul92 peptide bond.Isolated hexameric NCI 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.