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Mapping of the active site of recombinant human erythropoietin
S Elliott, T Lorenzini, D Chang, J Barzilay and E Delorme
Amgen Center, Thousand Oaks, CA 91320, USA.
Recombinant human erythropoietin (rHuEPO) variants have been constructed to
identify amino acid residues important for biological activity.
Immunoassays were used to determine the effect of each mutation on rHuEPO
folding. With this strategy, we could distinguish between mutations that
affected bioactivity directly and those that affected bioactivity because
the mutation altered rHuEPO conformation. Four regions were found to be
important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and
147 to 151. EPO variants could be divided into two groups according to the
differential effects on EPO receptor binding activity and in vitro biologic
activity. This suggests that rHuEPO has two separate receptor binding
sites. Mutations in basic residues reduced the biologic activity, whereas
mutations in acidic residues did not. This suggests that electrostatic
interactions between rHuEPO and the human EPO receptor may involve positive
charges on rHuEPO.
Volume 89,
Issue 2,
pp. 493-502,
01/15/1997
Copyright © 1997 by The American Society of Hematology
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