Covalent binding to α-macroglobulins of a protein with free SH groups produced by activated B cells: Blocking by d-penicillamine and gold compounds

Abstract

α 2-Macroglobulin (α 2M) complexed with proteinases or modified by the action of amines has been shown to affect immune responses in vitro, though as yet the mechanisms are poorly understood. Supernates from rabbit lymphoid cells cultured in medium with normal rabbit serum and 35S-methionine (or 14C-leucine) were found to contain intensely radiolabeled α-macroglobulins (αM) (α 1 and α 2) on electrophoresis. When human α 2M, instead of rabbit serum, was added to cultures, it also appeared radiolabeled, suggesting that lymphocyte-produced proteins (LyP) formed complexes with serum αM. These αM-associated LyP were produced in greater quantity when lymphocytes were cultured in the presence of mitogens; they were not produced by cells cultured in the presence of cycloheximide; they were produced primarily by B cells rather than T cells or macrophages. Pretreatment of serum or αM with methylamine, enhanced rather than inhibited the formation of LyP-αM complexes, a finding which is contrary to that expected if the LyP were a proteinase. Since this methylamine treatment of αM also results in the generation of free SH groups from the internal thioester bonds of αM, the formation of disulfide bonds between LyP and α M was considered. Indeed, (a) the LyP-αM complex formation was inhibited by N-ethylmaleimide, aurothiomalate, sodium aurothioglucose or d-penicillamine; (b) blocking the SH groups with NEM, of either culture fluid supernates or serum, had an inhibitory effect on the formation of these complexes; (c) the LyP-αM complexes were dissociated by sodium dodecyl sulfate (SDS) only after their reduction with 2-mercaptoethanol (2-ME). Thus, a disulfide bond was formed between αM and LyP with free SH groups (SH-LyP). Molecular sieving by high performance liquid chromatography (HPLC) of the serum-free radiolabeled supernates indicated that SH-LyP eluted at a position corresponding to a polypeptide of mol. wt of about 22,000. However, SDS-PAGE of the 22,000 mol. wt HPLC fraction showed that the major protein was approximately mol. wt 11,000 under both reducing and non-reducing conditions. In addition, the SH-LyP reduced by 2-ME from its binding site on α 2M had a mol. wt of about 11,000 in SDS-PAGE, suggesting that it was a non-covalent homodimer of mol. wt 11,000 polypeptides. We suggest that α 2M as well as SH-LyP may affect the immune system by functioning as SH-reactive agents.