myfavoriegene

This web page was made as an assignment for an undergraduate course at Davidson College.

My Favorite Protein:
Erythropoietin

Figure 1. Structure of EPO complexed with extracellular domainans of EPO receptor shown in ribbons. Image taken from PDI. Click on Image to find out more about its source. Permission Pending

Erythropoietin, also known as EPO is acidic glycoprotein growth factor that triggers erythrocyte, or red blood cell production (Erslev 1991). The 5 exons of the EPO gene encodes 193 amino acids, 27 of which are later cleaved off to produce a 166 amino acid long peptide although the circulating peptide contains 165 amino acids. The mechanism for this is cleavage is unknown (OMIM 2003). EPO is produced by the kidney or liver of adult mammals and also produced by the liver of fetal and neonatal mammals (Genecards 2003).

Erythropoietin triggers the production of erythrocytes that make up the majority of the cells within blood. The purpose of red blood cells is to transport respiratory gases. Low levels of oxygen levels in the body, known as hypoxia, causes the pathway leading to EPO production, and consequentially, erythrocyte production. This process is done through the use of the transcription factor, HIF-1 which many tissues give off in reduced oxygen conditions. HIF- 1 tells the kidney (or liver) to produce EPO. EPO then binds to two receptors (EPO- R) found on stem cells in the bone marrow of ribs, breastbone, pelvis and vertebrae. This leads to the maturation to functional red blood cells, and ultimately the increase of oxygen supply in tissues (Purves et al. 2001). Thus, when EPO is present, red blood cells mature. When EPO is unavailable, they die (Erslev 1991).

Figure 2. Figure from Life: The Science of Biology, Sixth Edition (Purves et al. 2001). This figure demostrates how low oxygen levels cause the growth factor HIF-1 to then trigger the kidney to make erythropoietin. EPO then causes stem cells to synthesize red blood cells which cause the oxygen supply within tissues to become greater. Permission Pending.

Erythropoietin is composed of an "up-up- down-down four helical bundle topology" and has "two small antiparallel beta strands typical of the short- chain class" (Syed et al. 1998). A disulphide bridge connects one of the pairs of antiparallel long helices from Cys 7 to Cys 161, while another antiparallel long helix (between alpha B and alpha C regions) is connected by a short loop. An irregularity at Gly 151 results in a kink in the alpha D helix. A beta sheet also results from amino acids of the AB and CD crossover loops. A, B, and C helices combined with many aromatic and hydrophobic regions form the interior of EPO. In addition, short helices exist near both alpha B and alpha C regions (Syed et al. 1998). EPO binds to two receptor proteins (EPObp2 and EPObp1), and thus has two binding sites (Syed et al. 1998).

Figure 4. Figure from Syed et al. 2003. Figure shows the Crystal structure of the erythropoietin complexed with its two receptors, EPObp2 and EPO bp1. Alpha helices are shown as cylinders while beta sheets are shown as ribbons. Permission Pending.

Since Gly 151 in the D helix of erythropoietin connects the side chain of Lys 152 into hydropobic contact with Val 63, Trp 51, and Phe 148 within the interior of the protein, the replacement of alanine at either position 151 or 152 would cause a loss of activity. Mutations to acidic amino acids do cause a considerable loss of reactivity although substitutions at the basic positions of Lys 20 and Lys 45 result in no loss of bioactivity. Two different amino acid positions that naturally contain Arg are very susceptible to mutations that result in loss of bioactivity. These two sites are Arg103 (that results in mutant R103A) and Arg 14 (that results in mutant R14Q). Both Arg 103 and Arg 14 are involved in site 2 binding, but a mutation in Arg 103 only results in loss of site 2 binding, whereas a mutation in Arg 14 results in an overall fivefold loss in affinity (for both binding sites 1 and 2) (Syed et al. 1998).

Table 1: Table from Nature (Syed et al.). This table shows the amino acid residues that are within the functional eptitope of erythropoietin. Mutations that will cause the most change in bioactivity are shown and the degree to which they cause loss of in vitro bioactivity is marked (bold and underlined, > 50 times; bold, >5 times; underlined, 2-5 times; unhighlighted, no effect).

The result of the underproduction of EPO is linked to a condition known as anemia, or the exhaustion of red blood cells (Purves et al. 2001). Among some of the diseases associated or coincide with underproduction of EPO are cancer, rheumatoid arthritis, HIV infection, sickle cell anemia, and anemia of prematurity. In some of these cases, like anemia of prematurity, a problem within the translation of the erythropoietin- coding gene into its protein is the cause of low EPO levels (Faruki and Kiss 1995). Anemia of prematurity seems to be caused by this underproduction of EPO. It is believed that the switch from the synthesis of erythropoietin in the liver to synthesis within the kidney that happens at birth in many mammals may be the cause of underproduction of EPO in premature infants. There is believed to be a delay in the switch to renal EPO synthesis, and so less erythropoietin is produced in premature babies. In other diseases, such as chronic renal disease, the decrease in EPO production is due to the fact that the kidney’s function is impaired, and likewise, because erythropoietin is produced mostly in the kidneys, its production is impaired also (Erslev 1991). However, in cases of anemia associated with cancer and other chronic diseases, the cause of decreased levels of EPO are due to the inhibition of EPO by inflammatory cytokines such as IL-1 and TNF that are generated in the presence of these diseases (Faruki and Kiss 1995).

Anemia caused by low levels of EPO can be treated through the use of recombinant EPO or rhu- EPO. The gene encoding EPO was abstracted, spliced into an expression vector and multiplied through the use of bacteria. Because people who have kidney failure undergo dialysis that removes toxins, and in the process EPO from their body, they lack whatever EPO their body did make. Recombinant EPO thus given to patients undergoing dialysis to restore their EPO levels (Purves et al. 2001)

NCBI. Nation Center for Biotechnology Information. Individual links found with Information.

Purves WK, Sadava D, Orians GH, Heller HC. 2001. Life: the Science of Biology, Sixth Edition. Sunderland, Massachusetts: Sinauer Associates, Inc, pp:324-325 and 879-881.

Syed RS, Reid SW, Li C, Cheetham JC, Aoki KH, Liu B, Zhan H, Osslund TD, Chirino AJ, Zhang J, Finer- Moore J, Elliott S, Sitney K, Katz BA, Matthews DJ, Wendoloski JJ, Egrie J, Stroud, RM. 1998. Effieciency of Signalling through cytokine receptors depends critically on receptor orientation. Nature 395: 511-516.