Conformational and sequential epitopes on the human granulocyte-colony stimulating factor molecule (hG-CSF) and their role in binding to human placenta receptors

JULIETA MARINO; AÍDA E. STERIN-PRYNC; CÉSAR H. CARBONETTO; LEONOR P ROGUIN

CYTOKINE.

ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD

Lugar: Amsterdam; Año: 2001 vol. 16 p. 41 - 50

1043-4666

Monoclonal antibodies (mAbs) named 8C2 and 6E3, directed against the recombinant humangranulocyte colony-stimulating factor (hG-CSF), were used as probes to study the cytokineorientation on its binding to receptors from human placenta. Competition enzyme linkedimmunoabsorbent assays (ELISA) revealed that mAb 8C2 would be directed to a linear epitope,whereas mAb 6E3 would delimit a more assembled epitope. Gel-filtration high performanceliquid chromatography (HPLC) of the immune complexes formed by incubating [125I]hG-CSFwith each mAb showed that epitope 8C2, but not 6E3, was altered after cytokine iodination. Inaddition, mAb 6E3 completely inhibited [125I]hG-CSF binding to human placental microsomes.Although [125I]mAb 6E3 was unable to bind to preformed hG-CSF?receptor complexes,[125I]mAb 8C2 did recognize hG-CSF previously bound to receptors, suggesting that epitope 8C2 would remain accessible in the hG-CSF?receptor complex. To identify the cytokine region defined by mAbs, hG-CSF was digested with different proteolytic enzymes: Arg-C, Glu-C, trypsin and chymotrypsin. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation. Results showed that mAb 6E3 would be directed to a conformation-dependent epitope located close to the hG-CSF binding domain and included into the sequence 1?122/123, whereas mAb 8C2 recognized the region 41?58, which represents a linear epitope left exposed after cytokine binding to receptors from human placenta.