Structural and Ligand Binding Analysis of Recombinant Blo t 13 Allergen from Blomia tropicalis Mite, a Fatty Acid Binding Protein

Abstract

Background: We have previously described a cDNA (clone Bt6) encoding a novel allergen from Blomia tropicalis, which showed sequence similarities to the FABP/P2/cellular retinoic acid binding protein/cellular retinol binding protein, a family of cytosolic lipid transport proteins (cLTPs). This work was planned to better characterize this allergen to which the official name Blo t 13 had been assigned. Methods: Fluorescence–based lipid ligand binding assays and secondary structure analysis by circular dicroism were carry out using recombinant Blo t 13 (rBlo t 13) protein. Structural predictions and molecular modelling were performed based on the amino acid sequence inferred from the open reading frame of Bt6 cDNA sequence. Results: rBlot t 13 binds the natural fluorescent fatty acid cis–parinaric acid and oleic acid by competition, but not retinol, retinoic acid, cholesterol, dansylated or anthroxylated fatty acids such as dansyl–DL–aminocaprylic acid and 12–(9–anthroyloxy)–stereate. Circular dichroism analysis indicated that rBlo t 13 comprises 45% β–sheet and 13% α–helix. The amino acid sequence of Blo t 13 modelled well to known crystal structures of cLTPs providing a tertiary structural model comprising ten β–strands organized into two β–sheets, and two short α–helices. Conclusion: Blo t 13 is a fatty acid–specific member of the β–rich cLTP family of proteins.