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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：

:T-cell-replacing factor (TRF)/IL-5 is a T-cell-derived glycoprotein which has pleiotropic activity on lymphoid and myeloid cells. 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). 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). A single amino acid sequence of each sample obtained beginning from methionine that was identical to that predicted from IL-5 cDNA. This finding supports the notion that secreted B151-TRF polypeptide consists of 113 amino acids. Purified B151-TRF supported eosinophilopoiesis of human bone marrow cells as effective as mouse rIL-5 and human rIL-5. B151-TRF competitively inhibited 35S-labeled rIL-5 binding to target cells to the same extent at rIL-5. 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. Reduction and alkylation of rIL-5 caused the loss of binding to its target cells. 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. 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.

journal_name

Mol Immunol

journal_title

Molecular immunology

authors

Takahashi T,Yamaguchi N,Mita S,Yamaguchi Y,Suda T,Tominaga A,Kikuchi Y,Miura Y,Takatsu K

doi

10.1016/0161-5890(90)90158-v

subject

Has Abstract

pub_date

1990-09-01 00:00:00

pages

911-20

issue

eissn

0161-5890

issn

1872-9142

journal_volume

pub_type

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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.