Domain structure of human α₂‐macroglobulin

Abstract

Digestion of methylamine‐treated α2‐macroglobulin (α2M·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 α2M·MA, and an 18 kDa fragment, constituting the carboxy‐terminal domain of α2M. This domain contained the receptor recognition site, exposed as a result of cleavage of the internal β‐cysteinyl‐γ‐glutamyl thiol esters in α2M. Compared with α2M‐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.

Expression and refolding of a high‐affinity receptor binding domain from rat α1‐macroglobulin

“…Half-maximal binding is observed at 12 and 8 nM, respectively. In contrast, the proteolytic RBD fragment isolated from ~2M-MA produces halfmaximal binding at approximately 100 nM [19][20][21]. The affinity of~M-MA is higher, consistent with the suggestion that tetrameric cqM-MA is able to bind to adjacent receptor molecules [10].…”

“…The determination of K~ was, however, based on an I%,lcm E 28o of 11.5. In contrast, we find that our longer fragment has l%,lcm anE 28o of 6.0.Thecalculatedvalue [31] 1~ l~m" ofE 280 is 5.2 for residues 1322 1477 (RBD), so the actual dissociation constant is likely to be higher than 40 nM, approaching that determined for human a~M-RBD [19][20][21]. The ~jM-RBDv expressed in this stu E~, km dy ( ~'80 determined to be 6.0) appears to be correctly folded since it, once purified and liberated from the fusion protein construct, appears monomeric in gel filtration under non-denaturing conditions.…”

“…138-residue Cterminal fragment can be released by limited proteolysis (RBD) [19][20][21]. The affinity of the ~2M-RBD fragment for ~2MR/LRP is approx.…”

Crystallisation and preliminary X‐ray analysis of the receptor‐binding domain of human and bovine α2‐macroglobulin

“…To cleave the thiol esters, human and bovine ~2M were treated with 200 mM methylamine and 20 mM iodoacetamide for 4 h [4]. The receptorbinding domains were obtained after cleavage of ~2M-MA with papain as described [18].…”

“…The Kd for binding of human and bovine RBD to c~2MR/ LRP, -60-125 nM in different systems [11,18,28], is much higher than the Ka for binding intact ~2M-MA (~40 pM and 2 nM for the high-and low-affinity binding, respectively) [21]. Therefore, it has been discussed whether RBD in its structure contains all information necessary for receptor recognition by c~2M [28,31].…”