Transferrins are extracellular proteins that bind iron. Vertebrate transferrins have well‐characterized roles in iron transport and immunity, but the functions of transferrins in most other animals are poorly understood. The goals of this study are to identify the functions of transferrin‐3 (Tsf3) from Drosophila melanogaster (fruit fly), and to determine whether Tsf3 is conserved in other species of insects. Our experimental approach has been to predict important features of the protein, analyze gene expression, perform RNAi‐mediated knockdown in cultured cells, and use phylogenetic analysis to identify Tsf3 orthologs. An analysis of the Tsf3 amino acid sequence predicts that Tsf3 is extracellular and anchored to the plasma membrane. Putative iron‐binding residues are present in the carboxyl‐lobe, but iron‐binding residues appear to be lacking in the amino‐lobe; therefore, we predict that only the carboxyl‐lobe binds iron. Tsf3 is expressed at a low level throughout development in many tissues, but it is very highly expressed in prepupal salivary glands. Cells of the prepupal salivary glands are exceptional because of their extensive DNA replication and because they synthesize large amounts of glue proteins that are used by the insect to attach itself to a solid surface prior to pupation. High expression of Tsf3 in the prepupal salivary glands suggests that Tsf3 may participate in the uptake of iron in these glands, possibly to be used as a cofactor for enzymes required for DNA synthesis, or perhaps to be used in the glue biosynthesis process. To test the hypothesis that Tsf3 is used for iron uptake by insect cells, we are in the process of evaluating the effect of RNAi‐mediated knockdown of Tsf3 on cellular iron content. Toward this goal, we verified that Tsf3 is expressed in cultured insect cells, synthesized dsRNA that targets Tsf3, and optimized a method to measure cellular iron content. Finally, we used a reciprocal BLAST method to search for possible orthologs of Tsf3 in insects from 12 different orders. We found that all 13 species analyzed have putative Tsf3 orthologs. Our findings demonstrate that Tsf3 is likely to be an extracellular, membrane‐bound, iron‐binding protein that may be involved in cellular iron uptake in diverse species of insects.Support or Funding InformationNSF IOS 1656388This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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