Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider

• Published in: Toxicon (Oxford) 2006-10, Vol.48 (5), p.509-519
• Main Authors: Felicori, L., Araujo, S.C., Machado de Ávila, R.A., Sanchez, E.F., Granier, C., Kalapothakis, E., Chávez-Olórtegui, C.
• Format: Article
• Language: English
• Subjects: Animal poisons toxicology. Antivenoms; Animals; Araneae; Biological and medical sciences; Epitope mapping; Epitopes - chemistry; Escherichia coli; Hemolysis - drug effects; Humans; In Vitro Techniques; Loxosceles intermedia; Loxosceles venom; Mass Spectrometry; Medical sciences; Mice; Molecular Weight; Necrosis - chemically induced; Necrosis - pathology; Phosphoric Diester Hydrolases - analysis; Phosphoric Diester Hydrolases - chemistry; Phosphoric Diester Hydrolases - immunology; Phosphoric Diester Hydrolases - toxicity; Platelet Aggregation - drug effects; Rabbits; Recombinant dermonecrotic protein; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Recombinant Proteins - toxicity; Serine Endopeptidases - chemistry; Serine Endopeptidases - immunology; Serine Endopeptidases - toxicity; Skin - drug effects; Skin - pathology; Spider Venoms - chemistry; Spider Venoms - immunology; Spider Venoms - toxicity; Synthetic peptides; Toxicology
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2006.06.019

In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8 mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32–35 kDa. The later result was confirmed by mass spectrometry (32,758 Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5 LD 50 of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.