Comparison of Alpha‐2‐Macroglobulins from Swine and Humans and their Copper Binding

Alpha‐2‐macroglobulin (a2M) is a member of the macroglobulin family of blood plasma proteins. It has a variety of functions, ranging from trapping proteases and transporting zinc to binding and transporting inflammatory and anti‐inflammatory cytokines and, growth factors and the small peptide regulating iron metabolism (hepcidin)[1]. a2M is the main form found in most mammals, while the alpha1‐inhibitor3 form dominates in the plasma of rodents. Some years ago, we determined in rats that a large plasma protein we named transcuprein was involved in transporting copper to the liver (exchanging copper with albumin), and upon purification found it was alpha1‐inhibitor3[2]. We then showed that human a2M also bound Cu(II) tightly, and that this copper was readily delivered by a2M to cultured human cells[3]. As we have been studying aspects of copper metabolism in pigs, we decided to also investigate the structure and copper binding of pig a2M to compare it to that of human a2M. Pig and human a2M were purified from heparinized Yorkshire pig plasma and from the heparinized plasma of human volunteers (under an approved university IRB protocol), using the established procedure for human a2M that uses a combination of PEG 8000 fractionation and Zn(II)‐immobilized metal affinity chromatography. The resulting samples were separated in large pore size exclusion chromatography on Sephacryl S300. Human a2M eluted had one large peak eluting with an Mr of ~900 kDa, and a much smaller peak of about 400 kDa. By SDS‐PAGE, both peaks showed the expected 180 kDa band, indicating that most of the human a2M was a tetramer, with a very small portion as a dimer. In the case of the pig, There was not tetrameric a2M; it was all in dimer form, and the subunits seen in SDS‐PAGE were two bands that together added up to ~180 kDa (120 + 60 kDa). Non‐denaturing PAGE gave single bands indicating that the smaller subunits of the pig a2M were combining into one structure. The copper content of the human a2M was 4 Cu atoms per tetramer (not taking into account potential non‐specific binding), but was 2 Cu atoms per tetramer when copper was first removed and added back in the presence of human or rat albumin. The Cu content of the pig a2M was 4 Cu atoms per dimer. Preliminary data obtained by EPR gave similar but not identical profiles and indicated there were two binding sites, the main one with Cu(II) atoms most likely bound to three Ns and one O. These findings indicate that in some species, a2M forms a dimer, while in others a tetramer; they bind Cu(II) in similar but not identical fashion; and that the subunits of pig a2M may have a vulnerable peptide bond that cleaves the 180 kDa unit into two pieces. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .