Bead based aptamer selection against transferrin for iron deficiency anemia

Transferrin is a glycoprotein produced by the liver, which regulates free iron concentrations in the body. (Crichton 1987) The body’s iron absorption areas include the duodenum, the first section in the small intestine, where dietary iron is found. Iron homeostasis is made possible through transferrin’s two binding sites, specific to Fe (III) for which its affinity is extremely high; although, it is quite sensitive to differentiations in pH. (Aisen 1978) When an apo-transferrin finds the iron in ferric form (Fe³⁺), it then becomes transferrin and then attaches to a transferrin receptor (TfR), found on the surface of the cell. The transferrin is hauled into the cell via receptor-mediated endocytosis. (Chen 2008) H⁺ATPases allow the protein to release its ions inside the cell. Once completed, the apo-transferrin is released through endocytic cycle to repeat the process. Because it is essentially taking up the free iron in the body, if there are high quantities of transferrin, then there are likely to be low levels of iron. A lack of iron correlates directly with oxygen transport system of hemoglobin throughout the body and iron deficiency anemia. (Alleyne 2008) Working with aptamers, I hope to find one that binds specifically and tightly to the target protein. An aptamer capable of inhibiting transferrin would be beneficial to persons with iron-deficiency anemia, in order to stop the transferrin from taking up the free iron in the body. Because the protein target has twobinding sites for iron it may be possible to develop an aptamer specific to those sites that way the free iron has no place to bind and will have to remain as free iron in the system. (See Figure 1.) Alternatively, if an aptamer is found to bind solely to the TfR, then that should indirectly inhibit the consumption of free iron in the system by apo-transferrin by making the transferrin serve no other function and thereby possibly forcing it to discontinue consumption of iron because TfR usually indicates iron deficiency. (See Figure 2.)

Figure 1-(Specific Aim 1) The iron attaches to apo-transferrin, thereby decreasing the amount of free iron in the body and making the molecule transferrin. An aptamer would need to be developed in order to take the place of the iron, allowing more free iron to be in the system.

Figure 2- (Specific Aim 2) The transferrin molecule binds to TfR, which should induce receptor-mediated endocytosis and ultimately remove free iron from the system and put them into various cells. An aptamer would need to be developed that would take the place of transferrin, hopefully eliminating the need to consume iron.

The target protein, transferrin, for Specific Aim 1, may be purchased through SunnyLab for $75 per 100ug. Its catalog number is P1-859and found at the following web address: http://www.sunnylab.com/product.php?productid=18031&cat=264&page=18. The target protein, apo-transferrin, for Specific Aim 2, may be purchased through R&D Systems for $95 per 100mg. Its catalog number is 3188-AT-100MG and found at the following web address: http://www.rndsystems.com/productresults.aspx?k=3188-AT.

References:

Chen, Chi-hong B. & et al. (2008).“Aptamer-based endocytosis of a lysosomal enzyme.” Proc Natl Acad Sci USA. 105(41):15908-13.

Skikne, B. S., Punnonen, K., Caldron, P. H., Bennett, M. T., Rehu, M., Gasior, G. H., Chamberlin, J. S., Sullivan, L. A., Bray, K. R. and Southwick, P. C. (2011). “Improved differential diagnosis of anemia of chronic disease and iron deficiency anemia: A prospective multicenter evaluation of soluble transferrin receptor and the sTfR/log ferritin index.”American Journal of Hematology. doi: 10.1002/ajh.22108

Alleyne M, Horne MK, Miller JL. (2008) “Individualized treatment for iron-deficiency anemia in adults.” Am J Med. 121:943-948.

Aisen, Phillip; Leibman, Adela; Zweier, Jay (1978). "Stoichiometric and Site Characteristics for Binding of Iron of Human Transferrin". Journal of Biological Chemistry 253 (6): 1930–1937.

Crichton R. and Charloteaux-Wauters, M. (1987). “Iron transport and storage.” Eur. J. Biochem. 164(3): 485-506.

*Will be undergoing only Specific Aim 2.

-by Vivian Renee Esparza

Here's a link to my full length proposal: http://dl.dropbox.com/u/36458548/UT369K-%20TfR%20Proposal.pdf

Here's a link to progress report: http://dl.dropbox.com/u/36458548/UT369K-%20Progress%20Report%201.docx

Here's a link to my final manuscript: http://dl.dropbox.com/u/36458548/Aptamer-Final%20Manuscript.docx