Conformational Changes of Loxosceles Venoms Sphingomyelinases Monitored by Dichroism Circular Spectroscopy: a biological implication

¹Andrade, S.A.; ¹Fernandes-Pedrosa, M.F.; ¹Gonçalves-de-Andrade, R.M.; ²Oliva, M.L.V.; ³van den Berg, C. W. and Tambourgi, D.V.

1 Laboratório de Imunoquímica, Instituto Butantan, SP, Brazil; 2 Laboratório de Bioquímica, Universidade Federal de São Paulo, SP, Brazil; ³University of Wales, College of Medicine, Cardiff, UK.

Envenomation by arachnids of the genus Loxosceles caninduce a variety of biological effects, including dermonecrosis and haemolysis. We have identified and characterized the toxins from L. intermedia venom that are responsible for all the local and systemic effects induced by whole venom. Two highly homologous proteins, termed P1 and P2, were purified to homogeneity and shown to be endowed with sphingomyelinase activity. The L. intermedia sphingomyelinases isoforms P1 and P2 show a high degree of identity with two other molecules in the spider venom. The latter molecules named P3 and P4, despite of high homology were ineffective in all activity assays. P1 and P2 toxins have been cloned and their DNA and protein sequences showed 57% of identity with other sphingomyelinase, termed H17, recently cloned and expressed from L. laeta venom gland. These recombinant proteins were endowed with all biological properties ascribed to the whole Loxosceles venoms. The aim of this study was to analyse the structure-function relationship of these sphingomyelinases. Circular Dichroism technique (CD) was used to determine the solution structure of the active sphingomyelinases and compare to the inactive isoform P3 from L. intermedia venom. Moreover, we have also evaluated the conformational changes of the active recombinant proteins induced by modifications of the pH and temperature conditions. At pH 7.4 and temperature of 37°C, recP1 showed to contain 32.1% of a helix and 16.2% of b-sheet; recP2 30.0% of a helix and 16.9% of b-sheet; H17 38.0% of a helix and 13.8% of b-sheet, while the inactive isoform P3 showed 22% of a helix and 18.9% of b-sheet. Extremely acidic or alkaline environments (pH 2.0 or 9.5) induced a considerable reduction of regular conformation of these recombinant proteins. In a similar study, but in different temperature values (37-55°C), the recombinant proteins were considerable more stable. Nevertheless, the CD spectroscopy showed a fairly loss of the regular conformation for these recombinant proteins when the temperature was increased to 80°C and abruptly reduced to 37°C. Dermonecrosis and haemolysis assays using recombinant proteins pre-treated in different pH and temperature conditions, showed that these conformational changes (observed by CD) were correlated directly to decrease of two principal clinical symptoms of the Loxoscelism.

Support: FAPESP, The Wellcome Trust, CNPq.

Correspondence to: sonia_andradebr@yahoo.com