ABSTRACT
Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-DL-alpha-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized beta-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a alpha-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of beta structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 degrees C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.
Subject(s)
Antigens, Helminth/chemistry , Onchocerca volvulus/chemistry , Retinol-Binding Proteins/chemistry , Amino Acid Sequence , Animals , Antigens, Helminth/genetics , Antigens, Helminth/metabolism , Binding, Competitive , Circular Dichroism , Dansyl Compounds/metabolism , Fatty Acids/metabolism , Fluorescent Dyes/metabolism , Molecular Sequence Data , Oleic Acid/metabolism , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/metabolism , Retinol-Binding Proteins/genetics , Retinol-Binding Proteins/metabolism , Sequence Alignment , Spectrometry, Fluorescence , Vitamin A/metabolismABSTRACT
A pool of sera from individuals classified as putatively immune (PI) to Onchocerca volvulus infection was employed in the screening of a fourth-stage larval cDNA expression library. A highly immunogenic clone, encoding the Ov 53/80 protein, was identified. The full length cDNA of clone 4.21 contained 2527 nucleotides encoding 769 amino acids of which 100 are glutamine residues (13%). Antibodies raised against recombinant protein encoded by a partial cDNA sequence (clone 73-k) recognized a 53 and 80 kDa protein in O. volvulus larval and adult parasite extracts, respectively. The antibodies localized the native protein in the cuticle, hypodermis, secretory vesicles and in granules of the glandular esophagus of larvae and in the hypodermis and the cuticle of adult worms. The recombinant 73-k polypeptide (r73) was recognized by 90-100% of sera from PI and infected individuals from Liberia, but only by 67% of similar groups from Ecuador. r73 specific IgG2 and IgG3 levels in the PI from Liberia and Ecuador, respectively, were significantly lower than in the infected, whereas the r73 specific IgG1/IgG3 or IgG1/IgG2 in the PI and the infected individuals from Liberia or Ecuador, respectively, were similar. The IgG4 specific antibody response in the PI from Liberia and Ecuador were lower than in the infected. The T-cell proliferative responses to r73 in infected individuals from Cameroon were found to be inversely correlated with their levels of microfilariae.
