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.
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.
Takahashi
T
T
Department of Biology, Kumamoto University Medical School, Japan.
Yamaguchi
N
N
Mita
S
S
Yamaguchi
Y
Y
Suda
T
T
Tominaga
A
A
Kikuchi
Y
Y
Miura
Y
Y
Takatsu
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002846:Chromatography, Affinity; D004220:Disulfides; D004804:Eosinophils / Q000166:cytology; D005260:Female; D015848:Interleukin-5 / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism
D004220:Disulfides; D015848:Interleukin-5; D046911:Macromolecular Substances; D011994:Recombinant Proteins