June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Erythropoietin R76E Possesses Reduced Erythropoietic Activity by Disruption of An Inter-Residue Salt Bridge
Author Affiliations & Notes
  • Wesley S Bond
    Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, TN
  • YPaul L Chang
    Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, TN
  • Tonia S Rex
    Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, TN
  • Footnotes
    Commercial Relationships Wesley Bond, None; YPaul Chang, None; Tonia Rex, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5462. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      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. doi: https://doi.org/.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×