Evolution of α2-macroglobulin. The structure of a protein homologous with human α2-macroglobulin from plaice (Pleuronectes platessa L.) plasma

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human α2-macroglobulin, and designated plaice α2-macroglobulin homologue or αMh, was shown to be a glycoprotein of s20,w 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice αMh migrated to the same position as half-molecules of human α2-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice αMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice αMh dissociated into subunits of Mr 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ‘mapping’ showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of αMh were cleaved into fragments of Mr about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the αMh with methylamine. [14C]Methylamine allowed to react with αMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human α2-macroglobulin. Reaction of αMh with a proteinase resulted in cleavage of a fragment of Mr 10000–15000 from subunit I. A proportion of the proteinase molecules trapped by αMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human α2-macroglobulin and plaice αMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.