Interleukin-4 and IL-10 Bind Covalently to Activated Human alpha 2 -Macroglobulin by a Mechanism That Requires Cys 949

Abstract

alpha ₂-Macroglobulin (alpha ₂M) functions as an extracellular carrier of diverse cytokines, including transforming growth factor-beta1 (TGF-beta1), that expresses anti-inflammatory activities. The results presented here demonstrate that interleukin-10 (IL-10) and IL-4, which also regulate the inflammatory response, bind to alpha ₂M. Unlike TGF-beta, IL-4 and IL-10 bind almost exclusively to the receptor-recognized, or activated, form of alpha ₂M. Purified IL-4-alpha ₂M complexes were predominantly covalent due to thiol disulfide exchange involving Cys⁹⁴⁹ in the alpha ₂M subunit. Blocking Cys⁹⁴⁹ with iodoacetamide significantly inhibited IL-4- and IL-10 binding. Bovine serum albumin(BSA), which possesses a free Cys residue and undergoes thiol disulfide exchange reactions, did not compete with alpha ₂M for the binding of IL-4 or IL-10. These results suggest a model in which IL-4 and IL-10 associate with activated alpha ₂M to form complexes that are initially noncovalent but unstable. In these complexes, Cys⁹⁴⁹ is properly aligned to undergo thiol disulfide exchange and generate stable, covalent IL-4- alpha ₂M and IL-10-alpha ₂M complexes.