BINDING AND INTERNALIZATION OF RECOMBINANT-HUMAN-ERYTHROPOIETIN IN MURINE ERYTHROID PRECURSOR CELLS

  • 1 May 1987
    • journal article
    • research article
    • Vol. 69  (5) , 1485-1490
Abstract
Erythropoietin (EPO) biosynthetically labelled with [35S]cystein was produced from Chinese hamster ovary (CHO) cells containing amplified copies of human EPO cDNA. The glycosylated recombinant [35S]EPO, purified to virtual radiochemical homogeneity, was biologically active. We studied the interaction of this labeled recombinant EPO with erythroid precursor cells from mice made anemic with phenylhydrazine. The [35S]-labeled molecule bound to erythroid precursors in a time- and temperature-dependent manner. The binding was specific for EPO, and neither insulin, transferrin, epidermal growth factor, nor multiplication stimulating activity could compete for EPO binding sites. In the presence of the 0.2% sodium azide, which blocks 80% to 90% of internalization, the recombinant molecule bound with an apparent Kd of 750 pmol/L and 100 t0 200 binding sites per cell at 37.degree. C. Asialo-EPO was a more effective competitor than sialated EPO for the available binding sites. Thus, the enhanced biological specific activity of asialo-EPO could result from its enhanced binding affinity. We also studied recombinant human EPO labeled with 125I and found that it also bound to the erythroid cells in a saturable and specific manner. After 90 minutes of incubation of 37.degree. C, most of the bound [35I]EPO was internalized, whereas most of the [125I]EPO remained on the cell surface. The reduced internalization of the iodinated molecule could account for the previously reported functional deficit associated with iodination.

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