Identification of funtional domains of the granulocyte-colony stimulating factor

MARINO JULIETA; STERIN-PRYNC A; ROGUIN L

San Carlos de Bariloche, provincia de Río Negro, Argentina

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular y XXXII Reunión Anual de Biofísica; 2003

Sociedad Argentina de Bioquímica y Biología Molecular y

The granulocyte-colony stimulating factor (G-CSF) is a glycoprotein involved in the proliferation and differentiation of neutrofilic precursors. In order to characterize functionally domains of the G-CSF molecule, monoclonal antibodies (mAbs 8C2 and 6E3) against the recombinant cytokine were employed. The identification of the antigenic regions recognized by mAbs was performed by determining the immunoreactivity of different peptides obtained either after G-CSF proteolytic digestion or by solid phase synthesis. Results showed that epitope 8C2 is formed by amino acids 39-52 and 155-164, whereas mAb 6E3 recognized sequences 1-22 and 94-123. In spite of defining distinct molecular regions, both mAbs significantly inhibited the proliferative response induced by G-CSF on a myeloid cell line. In addition, 125I-mAb 6E3 was unable to bind to G-CSF-receptor complexes preformed at 4°C, although 125I-mAb 8C2 recognized an exposed G-CSF region. To further explore mAb behavior, we examined the influence of temperature on the accessibility of epitope 8C2. When cytokine:receptor complexes were incubated at 37°C under conditions preventing the internalization, a significant reduction in the amount of accessible 8C2 epitopes was evident, suggesting that receptor aggregation could account for epitope masking. Taken together, our results indicate that whereas the neutralizing effect of mAb 6E3 would be due to the recognition of the G-CSF receptor-binding domain, mAb 8C2 would be inhibiting a receptor oligomerization process required for cytokine signalling.