The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.

No abstract

We have isolated and mapped the rHuEPO epitopes for three noncompeting anti-EPO monoclonal antibodies (MoAbs). The MoAb 9G8A recognizes a linear epitope that includes amino acids 13, 16, and 17. MoAb F12 recognizes a conformational epitope that includes amino acids 31 through 33, 86 through 91, and 138. MoAb D11 recognizes a conformational epitope that includes amino acids 64 through 78 and 99 through 110. MoAb D11 neutralizes rHuEPO activity which suggests that its epitope may contain the receptor binding domain. Analysis of the effect of mutations on folding allowed the identification of buried residues, alpha-helical, and non alpha-helical regions. This data along with epitope mapping data of anti rHuEPO monoclonals was used to model rHuEPO protein structure. A model consistent with the data is a 4-helix bundle with short and long interconnecting loops.

We have isolated and characterized three anti-recombinant human erythropoietin (rHuEPO) monoclonal antibodies (MoAbs) that recognize nonoverlapping epitopes on rHuEPO. Anti-EPO MoAb D11 neutralizes rHuEPO activity whereas MoAbs F12 and 9G8A do not. This suggests that D11 may bind to the rHuEPO active site. MoAbs F12 and D11 recognize conformation dependent epitopes whereas 9G8A does not. Immunoassays were developed for each monoclonal. The 9G8A immunoassay was novel and useful because immunoreactivity increased when rHuEPO was denatured. Disruption of disulfide bonds or removal of carbohydrate increased 9G8A immunoreactivity, which suggests that these elements are important for rHuEPO structure or stability.

No abstract

No abstract

No abstract

No abstract