Conformational and sequential epitopes on the human granulocyte-colony stimulating factor molecule (hG-CSF) and their role in binding to human placenta receptors.
Monoclonal antibodies (mAbs) named 8C2 and 6E3, directed against the recombinant human granulocyte colony-stimulating factor (hG-CSF), were used as probes to study the cytokine orientation on its binding to receptors from human placenta. Competition enzyme linked immunoabsorbent 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 performance liquid chromatography (HPLC) of the immune complexes formed by incubating [(125)I]hG-CSF with each mAb showed that epitope 8C2, but not 6E3, was altered after cytokine iodination. In addition, mAb 6E3 completely inhibited [(125)I]hG-CSF binding to human placental microsomes. Although [(125)I]mAb 6E3 was unable to bind to preformed hG-CSF-receptor complexes, [(125)I]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 alpha 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.
Marino
V J
VJ
Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Junin 956-1113 Buenos Aires, Argentina.
Sterin-Prync
A E
AE
Carbonetto
C H
CH
Roguin
L P
LP
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000936:Antigen-Antibody Complex / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism*; D011485:Protein Binding; D011863:Radioimmunoassay; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D007457:Iodine Radioisotopes; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D002918:Chymotrypsin; D014357:Trypsin