Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity.

J P Galizzi,B Castle,O Djossou,N Harada,H Cabrillat,S A Yahia,R Barrett,M Howard,J Banchereau

doi:10.1016/s0021-9258(19)40249-4

J P Galizzi, B Castle + Show 7 more

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https://doi.org/10.1016/s0021-9258(19)40249-4

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Abstract

The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.

Highlights

Jean-Pierre GalizziS$, Brian Castle%, Odile DjossouS, Nobuyuki HaradaV, H6lbne CabrillatS, Smina Ait YahiaS, Robin Barrettn, Maureen HowardlI, and Jacques BanchereauS
In order to facilitate the biochemical characterization of the interleukin 4 (IL-4) receptor (IL-4R), attempts have been made to up-regulate the expression of the cell surface IL-4R by culturing MLA 144 cells with phorbol myristate acetate, phytohemagglutinin, or concanavalin A
We describe the purification of a 130kDa glycoprotein isolated from the gibbon T cell line MLA 144 which binds human IL-4 with high affinity

Summary

Introduction

Jean-Pierre GalizziS$, Brian Castle%, Odile DjossouS, Nobuyuki HaradaV, H6lbne CabrillatS, Smina Ait YahiaS, Robin Barrettn, Maureen HowardlI, and Jacques BanchereauS. 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (&) similar to that measured in human cell lines Affinity cross-linking of ‘261-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately. It was recently shown to antagonize the interleukin 2-induced proliferation of normal [10, 11] and leukemic [12] B cells. It enhances the generation of cytotoxic T cells but inhibits the interleukin 2-dependent generation of lymphocyte-activated killer cells [13, 14]. It induces enriched B cell populations to produce IgE [15] and purified activated B cells to secrete IgG

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