EPIDERMAL GROWTH-FACTOR BINDING TO HUMAN ALPHA-2-MACROGLOBULIN - IMPLICATIONS FOR ALPHA-2-MACROGLOBULIN GROWTH-FACTOR INTERACTIONS

We have examined the binding of I-125-labeled human and mouse epidermal growth factors (EGF) to human alpha2-macroglobulin (alpha2M). In the presence of human neutrophil elastase, both mouse and human EGF bound to alpha2M, whereas little binding was found to native alpha2M. Binding was found to be predominantly covalent and mostly nonreducible by dithiothreitol. Greatly reduced binding was found when methylamine rather than proteinase was used to convert native alpha2M to fast-form alpha2M. Pretreatment of native alpha2M with either proteinase or methylamine greatly reduced binding of EGF. Titration of human I-125-EGF into native alpha2M, in the presence of 2 equiv of proteinase, gave a gradual increase in EGF binding as a function of EGF concentration. Between 0.8 and 1.0 equiv of hEGF were bound per alpha2M tetramer when 30 equiv of EGF were used. Reductive methylation of the alpha-amino group of mouse EGF eliminated most of the non-disulfide-mediated covalent binding. The pH dependence of binding of both mouse and human EGF to alpha2M was examined and showed more EGF bound at pH 6 than at pH 9. The reduction in binding with increasing pH was mostly for the covalent nonreducible component. These results suggest that EGF can react with the reactive thiol ester of proteinase-activated alpha2M by nucleophilic attack of the alpha-amino group and to a lesser extent by sulfide-disulfide exchange with the free SH of the cleaved thiol ester. The pH dependence is thought to result from competition with hydroxide for thiol ester cleavage. The low efficiency of incorporation of EGF found here, and similarly low efficiency of incorporation of insulin found by others, suggests that such covalent binding, although possible for many growth factors, is unlikely to be important in vivo as a means of efficiently complexing them to alpha2M. This contrasts with specific noncovalent binding of growth factors to alpha2M, such as appears to be the case for transforming growth factor beta.