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Wesley S Bond, YPaul L Chang, Tonia S Rex; Erythropoietin R76E Possesses Reduced Erythropoietic Activity by Disruption of An Inter-Residue Salt Bridge. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5462.
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© ARVO (1962-2015); The Authors (2016-present)
A mutated form of erythropoietin (EPO) at R76E is protective for photoreceptors and ganglion cells in multiple degeneration models and has reduced erythropoietic activity. We sought to determine whether a putative salt bridge interaction between R76 and E37 is necessary for erythropoietic activity of EPO. Within the E37-containing loop domain are residues necessary for binding to the high-affinity binding site of EPO receptor.
Missense mutations E37R and R76E were introduced into the human EPO gene by site-directed mutagenesis. Adult C57BL/6 mice were administered 1x109 vgc of rAAV2/1.CMV.EGFP, rAAV2/1.CMV.Epo-R76E, rAAV2/1.CMV.Epo-E37R, or rAAV2/1.CMV.Epo-E37R+R76E intramuscularly. At 1 week post-injection, blood was collected for evaluation of hematocrit.
Mice injected with rAAV2/1.CMV.EGFP or rAAV2/1.CMV.Epo-R76E had normal-range hematocrit levels of 43.3±2.4% or 49.8±7.8%, respectively. In contrast, mice treated with rAAV2/1.CMV.Epo-R76E+E37R or rAAV2/1.CMV.Epo-E37R had elevated hematocrit of 74.3±4.5% or 73.6±2.9%, respectively.
Restoration of the salt bridge by a reversed arginine-glutamate interaction by EPO-R76E+E37R produced higher hematocrit compared to EPO-R76E, suggesting that this interaction is necessary to facilitate erythropoietic activity of EPO potentially by stabilizing interaction with the high-affinity binding site of the EPO receptor. The E37R mutation alone also produced hematocrit elevation compared to EPO-R76E and similar elevation as EPO-R76E+E37R. This finding could be due to preservation of protein conformation by a novel interaction of the E37R residue with glutamate residues proximal to R76, such as E72.
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