Human α2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

Abstract

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.

Molecular Dissection of the Human α2-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling

“…Fig. 1 shows the ␣ 2 M sequences encoded by the fusion proteins studied here, and the relationship of these fusion proteins to the predicted domains in ␣ 2 M, as described by Doan and Gettins (19). The growth factor-binding site, expressed in FP3, is contained within the MG6 domain and the LRP-1 recognition site, which is encoded in FP6, is part of the MG8 domain.…”

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…Its mechanism of interaction with proteases has been described in detail (Sottrup-Jensen, 1989). Its covalent association with proteases sterically shields their active sites from large molecular substrates, hence permitting enzymatic hy-NEUTROPHIL SERINE PROTEASES drolysis of only small synthetic substrates (Doan and Gettins, 2007).…”

Functional Genomics of Tick Thioester-Containing Proteins Reveal the Ancient Origin of the Complement System

“…These proteins are synthesized as large precursors and become functional upon proteolytic cleavage of the highly variable segment located within the central part of the molecule [21] . The cleavage induces a rapid conformational change leading to the exposure of the reactive thioester (TE) bond and entrapping the proteases in a macromolecular cage.…”