Detection and characterization of metalloproteinases with gelatinolytic, fibronectinolytic and fibrinogenolytic activities in brown spider (Loxosceles intermedia) venom.
By studying Loxosceles intermedia (Brown spider) venom we were able to detect a proteolytic action on fibronectin and fibrinogen but an inability to degrade full length laminin, type I and type IV collagens. By studying enzyme inhibitors we observed that divalent metal chelators as EDTA and 1,10-phenanthroline completely blocked this cleaving action whereas serine-protease inhibitors, thiol-protease inhibitor and acid-protease inhibitor showed little or no effect on the proteolytic activity of the venom indicating involvement of a metalloproteinase. Zymogram analysis of venom detected a 35 kDa molecule with gelatinolytic activity. The metalloproteinase nature was further supported by its sensitivity to 4-aminophenyl mercuric acetate (APMA) treatment which decreased its molecular weight to 32 kDa, inhibition of its gelatinolytic effect by 1,10-phenanthroline and its elution from gelatin-sepharose affinity beads. In addition, zymogram experiments using fibronectin and fibrinogen as substrates detected a fibronectinolytic and fibrinogenolytic band at 28 kDa which changed its electrophoretic mobility to 20 kDa band after organomercurial treatment. The inhibitory effect of 1,10 phenanthroline and APMA sensitivity on this proteolytic effect confirmed the presence of a second metalloproteinase in the venom. The data presented herein describe two invertebrate metalloproteinases in L. intermedia venom with different specificities one gelatinolytic and another, fibronectinolytic and fibrinogenolytic, probably involved in the harmful effects of the venom.
Department of Cell Biology, Federal University of Parana, Jardim das Americas, Curitiba, Brazil.
L
L
Feitosa
W
W
Gremski
S S
SS
Veiga
M C
MC
Elias
E
E
Graner
O C
OC
Mangili
R R
RR
Brentani
D000818:Animals; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000032:analysis; D013111:Spider Venoms / Q000032:analysis* / Q000201:enzymology
D005353:Fibronectins; D013111:Spider Venoms; C055161:loxosceles venom; D005780:Gelatin; D005340:Fibrinogen; D010727:Phosphoric Diester Hydrolases; D008666:Metalloendopeptidases