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The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.

The complete amino acid sequence of the hen ovomacroglobulin (ovostatin) subunit has been determined from cDNA and partial peptide sequence analysis. Ovostatin is a tetrameric member of the α-macroglobulin (αM) family of proteins. The 4715 nt ovostatin cDNA encodes a 36- or a 16-residue signal peptide and a 1437-residue mature protein (162.2 kDa). At the protein level the overall score of sequence identity between ovostatin and mammalian αMs is 39-44%, indicating an early divergence from the line leading to the mammalian αMs. Ovostatin contains 56 mol glucosamine per mol subunit, and 12 of its Asn-residues are likely to be N-glycosylated. Including carbohydrate, the size of the ovostatin subunit is approx. 185 kDa. The ovostatin subunit is predicted to contain 12 intrachain disulfide bridges, and two subunits are predicted to be disulfide bound by two interchain bridges. One Cys residue may be unpaired or participate in dimer formation as a third interchain disulfide bridge. Ovostatin contains a unique 40-residue bait region. In contrast to other αMs, ovostatin contains no internal β-Cys-γ-Glu thiol ester, as a result of a Cys-to-Asn replacement (TGC or TGT to AAT), but the Gln-moiety of the thiol ester is preserved. By comparing the sequences of the receptor binding domain in αMs with the corresponding region of ovostatin possible determinants for receptor recognition of mammalian αMs are proposed.

Crystals of methylamine-treated α 2-macroglobulin (α 2M-MA), α 2-macroglobulin in complex with two molecules of trypsin, α 2M-T2, one molecule of plasmin, α 2-M-PL, and one molecule of plasmin followed by methylamine-treatment, α 2M-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of α 2M-MA diffracted to at least 9A˚resolution, crystals of α 2M-T2 diffracted to 10A˚resolution and crystals of α 2M-PL and α 2M-PL(MA) diffracted to 11A˚resolution. For α 2M-MA the cell parameters were determined as: a= b=257A˚, c=555A˚; and for α 2M-T2 as: a= b=247A˚, c=559A˚. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of α 2M-MA and α 2M-T2 contain one 360 kDa α 2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, Vm, was estimated at 5.6A˚ 3/Da. The crystal parameters of α 2M-PL and α 2M-PL(MA) were not determined.

Human β 2-glycoprotein (β 2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β 2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the β 2gpI cDNA. Upon isoelectric focusing, recombinant β 2gpI obtained from expression of β 2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β 2gpI isolated from plasma, and at least 5 polypeptides were visible

When α2-macroglobulin ( α2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other α-macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS-PAGE. While other α-macroglobulins extensively form crosslinks to the reacting proteinase, Limulusα2M does not. It rather becomes internally crosslinked. It was found from N-terminal sequence analysis of purified [ 14C]carboxymethylated peptides from Limulusα2M-trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln-1002 specifically to Lys-254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.

Digestion of methylamine-treated α 2-macroglobulin (α 2M·MA) with catalytic amounts of papain at pH 4.5 has been investigated. Cleavage of Lys(1313)-Glu resulted in two major products, which could be separated by gel chromatograhy: a large disulfide bridged fragment set nearly the size of intact α 2M·MA, and an 18 kDa fragment, constituting the carboxy-terminal domain of α 2M. This domain contained the receptor recognition site, exposed as a result of cleavage of the internal β-cysteinyl-γ-glutamyl thiol esters in α 2M. Compared with α 2M-trypsin complex the apparent affinity for binding to rat hepatocyte receptors was 0.1 and 2% at 4 and 37°C, respectively. The receptor-binding domain presumably forms a compact globular β-barrel-type structure, stable at pH 2.5–9.0. Chemical modification experiments suggest that receptor binding is contributed by a determinant formed by the precise folding of the polypeptide chain.

α 2-Macroglobulin (α 2M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacusα 2M molecule (Pα 2M) was radioactively labeled in the thiol ester structure with iodo [ 14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human α 2M and related proteins. The N-terminal sequence of Pα 2M was related to that recently determined for lobster α 2M [(1987) J. Biol. Chem. 262, 14606–14611].

The human placental receptor (α 2MR) for α 2-macroglobulin-proteinase complexes contains 3 polypeptides of approx. 500 kDa, 85 kDa, and 40 kDa. N-terminal sequence analysis of the 500 kDa and 85 kDa polypeptides, analysis of a random selection of peptides covering 536 residues from these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the α- and β-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119-4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769-1776). N-terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested that LRP is the functional receptor for α 2-macroglobulin-proteinase complexes (α 2MR) and in addition may have as yet unsettled functions in lipoprotein metabolism.

A recombinant version of the receptor binding domain (RBDv) of human α 2-macroglobulin (α 2M) has been expressed in E. coliand refolded using a novel iterative procedure. RBDv (Val 1299-Ala 1451) is extended by 15 residues at the N-terminal side of the Lys 1313-Glu papain cleavage site in human α 2M. RBDv contains the intra-chain bridge Cys 1329-Cys 1444and is soluble and monomeric. Competition experiments with 125I-labelled methylamine-treated α 2M reveal that RBDv binds to the placental receptor for transformed α 2M with a Kdof 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed α 2M to one receptor molecule.

The primary structure of bovine α 2-antiplasmin (α 2AP) has been determined from cDNA and partial peptide sequencing. Mature bovine α 2AP contains 470 residues and is 6 residues longer than human α 2AP. Alignment of the two protein sequences show that 81% of their amino acid residues are identically located. Bovine α 2AP has 5 N-linked carbohydrate groups, of which four are found in human α 2AP (AsnlOS, 274,288 and 295). Asn227 is the fifth carbohydrate attachement site in bovine α 2AP. The 3 Cys residues of bovine α 2AP are present as an unpaired residue (Cys131) and as a pair in a disulfide bridge (Cys49-Cys12). The assignment of the bridge in bovine α 2AP is at variance with the previous assignment of the two disulfide bridges in human α 2AP [Lijnen, H.R. et al. (1987) Eur. J. Biochem. 166, 565-574].

Bovine α 2-antiplasmin (α 2AP) has been purified and partially characterized. The amino acid composition is very similar to that of human α 2AP, and the N-terminal (23 residues determined) and reactive site loop sequences (42 residues determined) are highly homologous to those of the human protein. Compared with human α 2AP, bovine α 2AP has an 18-residue N-terminal extension, homologous with part of the pre-sequence of human α 2AP. A re-investigation of the N-terminal sequence of freshly prepared human α 2AP reveals a new form extended by 12 residues.

α2‐Macroglobulin (α2M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion‐exchange chromatography and gel filtration techniques. The Pacifastacusα2M molecule (Pα2M) was radioactively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C‐labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human α2M and related proteins. The N‐terminal sequence of Pα2M was related to that recently determined for lobster α2M [(1987) J. Biol. Chem. 262, 14606–14611].

Uptake of rat and human α 2-macroglobulin-trypsin complexes was measured in rat hepatocytes, rat and human adipocytes and human fibroblasts. Uptake and degradation of 125I-labelled rat complex were about one-third of that of the human complex in the various isolated cell types. In rat hepatocytes, the apparent Kmfor cell association of the rat complex was about 16 nM as compared to about 6 nM for the human complex. The Vmaxvalues were similar, about 1 × 10 4molecules · cell −1· min −1. Thus, rat α 2-macroglobulin (an acute-phase protein) complexed with trypsin follows the same pathways of uptake as the human homologue, although with a somewhat lower affinity for the uptake system.