‘Partitagin’ a hemorrhagic metalloprotease from Hippasa partita spider venom: Role in tissue necrosis

Abstract

The poisonous bite by Hippasa partita, a funnel web spider from the Indian subcontinent has been demonstrated to give rise to severe dermo- and myonecrosis. In this work a hemorrhagic metalloprotease, Partitagin was purified from H. partita venom by successive chromatography on Sephadex G-100, DEAE Sephadex A-50 and Biosep DEAE columns. SDS-PAGE, reversed phase HPLC on a C 4 column, N-terminal amino acid sequencing and MALDI-TOF mass spectrometry confirmed the homogeneity. Partitagin was assayed using fat free casein as substrate. EDTA, 1,10-phenanthroline and cyanide, inactivated it irreversibly while, EGTA, PMSF, leupeptin, pepstatin and aprotinin did not inhibit. The presence of Zn +2 was confirmed by atomic absorption spectrometry. Partitagin caused hemorrhage when tested in a mouse model. Light microscopy of skin tissue sections at the site of injection revealed extensive damage of extracellular matrix (ECM) in which the basement membrane surrounding blood vessels and capillaries showing signs of extensive destruction and also loss of vessel wall integrity. Similar intense damage was also noticed in the ECM of muscle tissue sections but with no damage caused to myocytes. Partitagin showed specificity of action on the components of ECM and degraded collagen type-IV and fibronectin but not collagen type-I. Partitagin was devoid of edema, myotoxicity and lethality. This is the first report on the isolation and characterization of a toxin from spider venom in the Indian subcontinent.