PMID: 2215480

 

    Legend: Sugar

Title : Structural comparison of murine T-cell (B151K12)-derived T-cell-replacing factor ( IL-5 ) with rIL-5 : dimer formation is essential for the expression of biological activity

Abstract :
  1. T-cell-replacing factor ( TRF )/ IL-5 is a T-cell-derived glycoprotein which has pleiotropic activity on lymphoid and myeloid cells
  2. IL-5 polypeptide translated into Xenopus oocytes are heterogeneous in molecular size (40,000 to 60,000 under nonreducing conditions) and yields a monomeric form (Mr of 25,000 to 30,000) under reducing conditions (J. Immun., 140, 1175-1181, 1988)
  3. We purified T-cell-derived TRF and rIL-5 using anti- TRF / IL-5 antibody-coupled affinity column from supernatants of a T-cell hybridoma B151K12 and supernatants of HeLa cells, respectively, which had been transfected with murine IL-5 cDNA, and determined their partial N-terminal amino acid sequence (27 residues for B151- TRF and 13 residues for rIL-5 )
  4. A single amino acid sequence of each sample obtained beginning from methionine that was identical to that predicted from IL-5 cDNA
  5. This finding supports the notion that secreted B151- TRF polypeptide consists of 113 amino acids
  6. Purified B151- TRF supported eosinophilopoiesis of human bone marrow cells as effective as mouse rIL-5 and human rIL-5
  7. B151- TRF competitively inhibited 35S-labeled rIL-5 binding to target cells to the same extent at rIL-5
  8. Treatment of purified rIL-5 and B151- TRF with reducing reagents such as 2-ME, sodium borohydride or dithiothreitol produced a monomeric form of IL-5 which did not exert a biological activity
  9. Reduction and alkylation of rIL-5 caused the loss of binding to its target cells
  10. These results strongly suggest that B151- TRF exists as a homodimer and its primary structure and secondary structures are identical to those of rIL-5
  11. Moreover, the formation of inter-molecular disulfide bond(s) linked by two pairs of cystein residues is essential for the expression of the biological activity of mouse IL-5