Structural and ligand binding analysis of recombinant Blo t 13 allergen from Blomia tropicalis mite, a fatty acid binding protein

Background: We have previously described a cDNA (clone Bt6) encoding a nove l allergen from Blomia tropicalis, which showed sequence similarities to th e FABP/P2/cellular retinoic acid binding protein/cellular retinol binding p rotein, a family of cytosolic lipid transport proteins (cLTPs). This work w as planned to better characterize this allergen to which the official name Blo t 13 had been assigned. Methods: Fluorescence-based lipid ligand bindin g assays and secondary structure analysis by circular dicroism were carry o ut using recombinant Blo t 13(rBlo t 13) protein. Structural predictions an d molecular modelling were performed based on the amino acid sequence infer red from the open reading frame of Bt6 cDNA sequence. Results: rBlot t 13 b inds the natural fluorescent fatty acid cis-parinaric acid and oleic acid b y competition, but not retinol, retinoic acid, cholesterol, dansylated or a nthroxylated fatty acids such as dansyl-DL-aminocaprylic acid a nd 12-(9-an throyloxy)-stereate. Circular dichroism analysis indicated that rBlo t 13 c omprises 45% beta-sheet and 13% alpha-helix. The amino acid sequence of Blo t 13 modelled well to known crystal structures of cLTPs providing a tertia ry structural model comprising ten beta-strands organized into two beta-she ets, and two short alpha-helices. Conclusion: Blo t 13 is a fatty acid-spec ific member of the beta-rich cLTP family of proteins.