A comparison of the sequence of the subunit of human a2-macroglobulin (a2M; 1451 amino acid residues) with that of murine complement component pro-C3 (1639 amino acid residues) reveals eight extended regions of sequence similarity. These regions contain between 19% and 31% identically placed residues and account for 75% and 67%, respectively, of the polypeptide chains of a2M and pro-C3. Published sequence data for complement component C4 show that segments of this protein match well with corresponding stretches in a2M and pro-C3. It is proposed that a2M, C3, and C4, which all contain a unique activatable j-cysteinyl-y-glutamyl thiol ester, have a common evolutionary origin and are homologous proteins. Several larger regions of low sequence similarity indicate the presence of structural domains in each of these proteins that specifically modify an underlying common gross structure. The quartets of basic residues in pro-C3 and pro-C4, at which cleavage takes place to produce the mature subunits of these proteins, and most of the residues forming the anaphylatoxin peptides of C3 and C4 (C3a and C4a) are absent in a2M. In addition, C3 and C4 contain large portions, which extend beyond the COOH terminus of a2M.Among the plasma proteins, a2-macroglobulin (a2M) and the complement components C3 and C4 share a unique sensitivity toward small nitrogen nucleophiles such as hydrazine, hydroxylamine, and methylamine. Upon incubation with these compounds, the proteinase binding properties of a2M and the hemolytic activity of C3 and C4 are gradually lost (1-3). It was recently shown that a2M, C3, and C4 have several other features in common: a characteristic pattern of heat/ denaturation cleavage (4-9); an ability to incorporate methylamine, thereby forming a residue of y-glutamylmethylamide (10-13); an appearance of SH groups after incubation with nitrogen nucleophiles or chaotropes (8, 14-19) or after activation with proteolytic enzymes (8,14,16,18); and an ability of the proteolytically activated proteins to participate in covalent binding reactions (20-23), engaging the methylamine reactive Glx residue (24-27). These features result from the presence of a unique type of reactive site in each of these proteins, identified as an activatable internal B-cysteinyl-y-glutamyl thiol ester (13,14,16), located in the achain part of C3 and C4 (13,24,28,29) and in each of the four identical subunits of a2M (10,17,30). Since the thiol ester structure, -Gly-Cys-Gly-Glu-Glx-, is found in regions LIC= of conserved amino acid sequence in all three proteins (10,13,17,24,(28)(29)(30), an evolutionary relationship is apparent. This is further substantiated by the similar size of the a2M subunits [Mr, 180,000 (31,32)] and the single chains of the pro-C3 and pro-C4 molecules [Mr, 175,000 and 190,000,], and by the organization of the individual chains of C3 and C4 within pro-C3 and pro-C4 [,-a and 3,S-a y, respectively (36-39)]. The high degree of sequence homology between the anaphylatoxins generated from C3 and C4 upon proteolytic act...

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The α2M tetramer of Mr 725,000 consists of four apparently identical single polypeptide chain monomers of Mr 180,000, corresponding to about 1450 amino acid residues. At the present stage of our sequence determination 1383 residues can be accounted for in three segments of continuous sequence namely the N-terminal (residues 1-440); the “middle” of 298; the C-terminal of approx. 645 residues. The eight oligosaccharide prosthetic groups are glucosamine-based and attached to Asn-residues, five in the N- terminal, three in the C-terminal segment. 25 CNBr-frag- ments have been found, 22-Met-residues have been overlapped. 23 Cys-sequences have been determined so far and ten -S-S- bridges identified. The Cys-residue (approx, no. 472 from the C-terminal in each chain) of the sequence is thiol-ester linked to the γ-carboxyl of the Glx-residue in native α2M which has not been reacted with proteinase or “inactivated” with methylamine or by heat inactivation.Neither extensive internal sequence homology, nor homology with other proteinase inhibitors of known primary structure has yet been observed.

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