title: Two new phospholipase D isoforms of Loxosceles laeta: cloning, heterologous expression, functional characterization, and potential biotechnological application.
abstract: Toxin phospholipases-D present in the venom of Loxosceles spiders is the principal responsible for local and systemic effects observed in the loxoscelism. In this study, we describe the cloning, expression, functional evaluation, and potential biotechnological application of cDNAs, which code for two new phospholipase D isoforms, LIPLD1 and LIPLD2, of the spider Loxosceles laeta. The recombinant protein rLIPLD1 had hydrolytic activity on sphingomyelin and in vitro hemolytic activity on human red blood cells, whereas rLIPLD2 was inactive. The purified recombinant proteins and the venom are recognized by polyclonal anti-rLIPLD1 and rLIPLD2 sera produced in animals and conferred immunoprotection against the venom. These new isoforms reinforce the importance of the multigene family of phospholipases-D present in Loxosceles spiders. A highly immunogenic inactive isoform such as rLIPLD2 raises important expectation for its use as a potential immunogenic inducer of the immunoprotective response to the toxic action of the venom of Loxosceles laeta.
authors.affiliation: Laboratory of Molecular Parasitology, Department of Medical Technology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 160, Antofagasta, Chile.
authors.forename: A
authors.initials: A
authors.lastname: Catalán
authors.forename: W
authors.initials: W
authors.lastname: Cortes
authors.forename: H
authors.initials: H
authors.lastname: Sagua
authors.forename: J
authors.initials: J
authors.lastname: González
authors.forename: J E
authors.initials: JE
authors.lastname: Araya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000633:toxicity; D010802:Phylogeny; D016415:Sequence Alignment; D013045:Species Specificity; D013111:Spider Venoms / Q000201:enzymology* / Q000633:toxicity; D013112:Spiders / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D