Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.
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 25.8mg 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 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). 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 2.5LD(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.
Felicori
L
L
Departamentos de Bioquímica-Imunologia, Universidade Federal de Minas Gerais, CEP 30161-970, Belo Horizonte, MG, Brazil.
Araujo
S C
SC
de Avila
R A Machado
RA
Sanchez
E F
EF
Granier
C
C
Kalapothakis
E
E
Chávez-Olórtegui
C
C
D000818:Animals; D000939:Epitopes / Q000737:chemistry; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D051379:Mice; D008970:Molecular Weight; D009336:Necrosis / Q000139:chemically induced / Q000473:pathology; D010727:Phosphoric Diester Hydrolases / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D010974:Platelet Aggregation / Q000187:drug effects; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D012867:Skin / Q000187:drug effects* / Q000473:pathology; D013111:Spider Venoms / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*
D000939:Epitopes; D011994:Recombinant Proteins; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D; D012697:Serine Endopeptidases