Name: Pedro Felipe Malacarne
Type: MSc dissertation
Publication date: 30/03/2017

Namesort descending Role
Rita Gomes Wanderley Pires Advisor *

Examining board:

Namesort descending Role
Juliana Brambilla Carnielli Trindade External Examiner *
Marco Cesar Cunegundes Guimarães Internal Examiner *
Rita Gomes Wanderley Pires Advisor *
Suely Gomes de Figueiredo Co advisor *

Summary: Cytolysins are substances elaborated by many different organisms that exhibit
toxic activities on cells, causing its dissolution through cytolysis. Fish venom
cytolysins are multifunctional proteins that display neurotoxic, cardiotoxic and
inflammatory effects, described as "protein lethal factors". A pore-forming
labile hemolytic cytolysin called Sp-CTx (Scorpaena plumieri - Cytolytic Toxin)
(glycoprotein, 2 subunits of 65 kDa) was recently purified from the venom of
the scorpionfish Scorpaena plumieri. The aim of this work was to establish
optimal storage conditions to maintain the activities of this toxin and to
continue its biochemical and pharmacological characterization. For this, SpV
(Scorpaena plumieri Venom) and Sp-CTx were stored in different conditions
(pH, temperature and in the presence of co-solutes), for different periods of
time. Afterwards, their hemolytic activity on rabbit erythrocytes was compared
to the activity of the freshly extracted venom and purified toxin. We also
evaluated the effects of different cations and membrane lipids on the
hemolytic activity of Sp-CTx. It was demonstrated that storage of Sp-CTx at -4
ºC in pH 7,4 with 500mM NaCl, 1M (NH4)2SO4, 60mM of threalose and 10%
glycerol, and at -80 and -196 ºC with glycerol 10% were effective in
maintaining the hemolytic activity. Furthermore, the hemolytic activity is
calcium dependent and completely inhibited by EDTA. Cholesterol and
phosphatidylglycerol reduced the hemolytic activity of Sp-CTx which suggests
that it is a barrel-pore-forming toxin and that the pore formation is dependent
of electrostatic interactions with membrane lipids. Through western blot, using
the “DIG Glycan Differentiation” kit, sugar motifs of Sp-CTx were evaluated,
which suggested the presence of: galactose (1-4) N-acetylglucosamine and
sialic-acid (2-3) galactose in N- and/or O- glycan complexes. Using the mouse
hindpaw test it was shown that Sp-CTx induced intensive nociception and
dose-dependent edematogenic responses in the mice footpad Sp-CTx. The
local induced edema was considerably reduced by previous administration of
aprotinin and HOE-140. These data suggest the involvement of the kinin–
kallikrein system in the inflammatory response induced by Sp-CTx.
Keywords: Scorpionfish, Cytolysin, Venom and Stability

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