Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom |
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Authors: | Daniele Chaves‐Moreira Olga M Chaim Youssef B Sade Kátia S Paludo Luiza H Gremski Lucélia Donatti Juliana de Moura Oldemir C Mangili Waldemiro Gremski Rafael B da Silveira Andrea Senff‐Ribeiro Silvio S Veiga |
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Institution: | 1. Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil;2. Department of Biochemistry, Federal University of S?o Paulo, S?o Paulo, Brazil;3. Medical Clinic Discipline, Department of Medicine, Federal University of S?o Paulo, S?o Paulo, Brazil;4. Department of Basic Pathology, Federal University of Paraná, Curitiba, Paraná, Brazil;5. Department of Physiology, Federal University of Paraná, Curitiba, Paraná, Brazil;6. Catholic University of Paraná, Health and Biological Sciences Institute, Curitiba, Paraná, Brazil;7. Department of Structural, Molecular Biology and Genetics, State University of Ponta Grossa, Ponta Grossa, Paraná, Brazil |
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Abstract: | Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc. |
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Keywords: | brown spider venom phospholipase‐D catalysis hemolysis |
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