Biochemical properties and physiological functions of extracellular iron-binding proteins

Abstract

Iron is an essential micronutrient to all animals, but it is highly toxic if it is not properly managed. Insects have processes to maintain iron homeostasis through iron absorption, transport, and storage; moreover, as part of their innate immunity, insects produce iron sequestering proteins to protect against iron-scavenging pathogens. Transferrins are a family of iron-binding proteins found in animals. In mammals, secreted transferrins reversibly bind two ferric ions and function in iron transport and immune-related iron sequestration. Insect iron homeostasis is poorly understood, and the role of insect transferrin in iron homeostasis is not well-studied. This dissertation explores the biochemical and structural characteristics and in vivo localization of secreted insect transferrin-1 (Tsf1) using two model insect species, Drosophila melanogaster and Manduca sexta. Biochemical characterization of Tsf1 was performed to test the hypotheses that the protein binds iron with high affinity and releases the iron as a function of pH decrease. Experiments revealed that Tsf1 binds a single ferric ion with high affinity at neutral pH (log K’ = 18), and releases iron over a moderately acidic pH range (6.2 to 5.0). These results indicated that Tsf1 may have physiological functions similar to some mammalian transferrins: binding and sequestering iron in extracellular fluids and releasing iron in the acidic environment of endosomes for uptake into cells. Structural characterization of Tsf1 revealed a novel mechanism of iron coordination, where iron was ligated by two tyrosine residues and two carbonate anions. Mutational analysis of Tsf1 explored the importance of the two tyrosines and a highly conserved carbonate-binding asparagine to the properties of high-affinity iron binding and pH-mediated iron release. In vivo studies of Tsf1 localization in D. melanogaster tissues were performed to test the hypothesis that Tsf1 is endocytosed into cells as a possible mechanism for iron transport. Except for two specialized cell types, Tsf1 did not localize in the endosomes of insect cells, which indicated that uptake of Tsf1 is not an essential mechanism for iron transport in healthy insects. This work furthers our understanding of Tsf1 function in iron sequestration and transport in insects, and it provides insight regarding the similarities and differences in iron homeostasis between insects and humans.