Biochemical study of a recombinant soluble interleukin-2 receptor. Evidence for a homodimeric structure.
A truncated soluble form of the human interleukin-2 receptor p55 chain (T-S-IL-2R) was expressed to high levels in RODENT (mammalian) cells and affinity-purified. Its biochemical behavior was analyzed by polyacrylamide gel electrophoresis (PAGE), gel filtration, and sucrose gradient centrifugation. It migrated as a single 40-kDa band on sodium dodecyl sulfate-PAGE (reducing or nonreducing conditions), whereas it ran as a 80-kDa component on native PAGE or as a 86-kDa component on gel filtration. The combination of gel filtration and density gradient sedimentation gave a Stokes radius of 4.0 nm and a sedimentation coefficient of 3.72 S. The deduced molecular mass was 67 kDa, and the fractional ratio was 1.516. These data therefore indicated that the T-S-IL-2R was secreted as an homodimer of two noncovalently associated 40-kDa subunits. Cross-linking experiments using bifunctional reagents enabled the materialization of the dimeric structure on sodium dodecyl sulfate-PAGE. Stoichiometric binding studies using two monoclonal antibodies (mAbs 33B3.1 and 11H2) reacting with separate epitopes on the p55 chain also strongly supported the dimeric structure. Indeed, there was one binding site for the 33B3.1 mAb (and Fab fragment) per T-S-IL-2R 40-kDa subunit, whereas the 11H2 mAb (or Fab fragment) could bind only half a site per subunit, a result which could only be explained when assuming more than one subunit for the native T-S-IL-2R. Soluble interleukin-2 receptor species were also purified from culture supernatants of either L cells transfected with the full-length p55 cDNA or a normal alloreactive T cell clone. Similar biochemical behavior and reactivities with the two mAbs were found. Finally, cell-surface p55 chains expressed either by pgL21 or 4AS cells bound the 33B3.1 and 11H2 mAbs in a 2:1 ratio, suggesting that the p55 chains are also associated as homodimers when imbedded in the membrane.
Jacques
Y
Y
Institut National de la Santé et de la Recherche Médicale U211, Unité de Recherche sur les Effecteurs Lymphocytaires T, Nantes, France.
Le Mauff
B
B
Godard
A
A
Naulet
J
J
Concino
M
M
Marsh
H
H
Ip
S
S
Soulillou
J P
JP
D000818:Animals; D000911:Antibodies, Monoclonal; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D007739:L Cells / Q000276:immunology; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D015375:Receptors, Interleukin-2 / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014162:Transfection
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins