Orientation of the human granulocyte-colony stimulating factor molecule (hG-CSF) in its binding to receptors

VERÓNICA J. MARINO; AÍDA STERIN-PRYNC; ALEJANDRO VIDAL; LILIA A. RETEGUI; LEONOR ROGUIN

Punta del Este, V Congreso Latinoamericano de Inmunología

Congreso; V Congreso Latinoamericano de Inmunología.; 1999

   The hG-CSF is a 174 amino-acid glycoprotein responsible for the proliferation, differentiation and activation of  hematopoietic cells of the neutrophilic granulocyte lineage. In order to characterize the interaction of hG-CSF to its receptors we have employed different monoclonal antibodies (mAbs) obtained after immunization of mice with recombinant hG-CSF. ELISA experiments revealed that one of these mAbs, named 8C2, recognized both the reduced and alkylated cytokine as well as the native hG-CSF, suggesting that the mAb did not recognize a conformational epitope. It was also demonstrated that mAb 8C2 was unable to inhibit the cytokine-induced proliferation of NFS-60 cells, a murine leukemia cell line. Furthermore, this mAb would be directed to a region left exposed in the G-CSF:receptor complex since 125I-mAb 8C2 recognized hG-CSF previously bound to receptors from human placenta. To identify the cytokine region defined by the mAb, hG-CSF was digested with different proteolytic enzymes: Arg-C, Glu-C and chymotrypsin. Immunoreactivity of the resulting peptides was examined by Western Blot and their sequences were established by Edman degradation. Data obtained indicated that the smallest immunoreactive region recognized by this mAb consisted of residues 23-58. Thus, our results suggested that the 23-58 sequence would contain a sequential epitope that remains exposed in the cytokine:receptor complex and indicate that the 36-42 disulfide bridge included in this fragment would not be required for G-CSG:mAb interaction.