Biochemical Characterization of Two Insect Serum Transferrins
Authors: Weber Jacob , Brummett Lisa , Geisbrecht Brian , Kanost Michael , Gorman Maureen 
Published: June 2021

Proper iron homeostasis is crucial for the overall health of nearly all organisms. One of the key factors in facilitating iron regulation is the family of proteins known as transferrins. Vertebrate transferrins are known for their ability to bind one ferric iron ion in each of their two lobes (N‐and C‐lobe). Binding occurs with high affinity in the blood and other fluids, and transferrin‐bound iron is safely delivered into cells via endocytosis. These functions help to protect against oxidative stress, supply and maintain iron levels, and sequester iron from invading pathogens. The details of transferrin's structure, iron binding residues and cellular uptake mechanism have all been established for vertebrates; however, details have yet to be ironed out for invertebrates. For most insect transferrins, amino acid sequence alignments suggest a similar overall size and structure, but iron binding in only the N‐lobe. Moreover, a putative substitution of the iron‐coordinating histidine residue is found in nearly all insects. These differences in invertebrate transferrins are puzzling and lead to questions about their functionality. As methods for safe and effective pest control strategies continue to improve, it is important to understand the fundamentals of how insect systems work and differ from vertebrates. Our research aims to determine the function(s) of insect transferrin via biochemical and structural analysis, using Drosophila melanogaster and Manduca sexta transferrin (DmTsf1 and MsTsf) as models. We purified DmTsf1 from a baculovirus expression system and MsTsf1 from larval hemolymph. Both proteins were purified by using ammonium sulfate precipitation and a series of column chromatography steps. We showed that apo‐transferrin but not holo‐transferrin can inhibit Escherichia coli growth, suggesting an immune function that relies on iron sequestration. Using equilibrium dialysis, we determined the apparent dissociation constant of apo‐DmTsf1 for iron is 10−18 M (at pH 7.4). Furthermore, these data demonstrated there is only one iron‐binding site. DmTsf1's affinity is less but comparable to mammalian transferrin's affinity for iron, Kd = 10−22 M, suggesting a role in iron sequestration. Analysis of absorbance spectra using different concentrations of metal show that apo‐DmTsf1 can also bind one molar equivalent of copper or zinc ions. Affinity measurements for MsTsf are still underway. Failed attempts to crystallize deglycosylated holo‐DmTsf1 and holo‐MsTsf for X‐ray crystallographic analysis of structure, has led us to express and purify the single N‐lobe of DmTsf1. Once the purification is complete, we will attempt to crystalize the N‐lobe and obtain a structure. With this information, we can identify the iron‐binding residues and compare the novel insect transferrin structure to the many vertebrate structures. We are hopeful that a structure will provide further insight into the functions of insect transferrins. Further biochemical pursuits into the function of insect transferrins will include determining the role of the C‐lobe and how iron from insect transferrins is delivered to cells.Support or Funding InformationNSF: 1656388NIH: R37 GM041247This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.