Further characterization of the covalent linking reaction of α2-macroglobulin

Abstract

It is shown that non-proteolytic proteins can become covalently linked to alpha 2M (alpha 2-macroglobulin) during its reaction with proteinases, and that this probably occurs by the mechanism that leads to the covalent linking of proteinases described previously [Salvesen & Barrett (1980) Biochem. J. 187, 695-701]. The covalent linking of trypsin was at least partly dependent on the presence of unblocked lysine side chains on the protein. The covalent linking of proteinases was inhibited by nucleophiles of low Mr, and these compounds were themselves linked to alpha 2M in a molar ratio approaching one per quarter subunit. Peptide "mapping" indicated that the site of proteinase-mediated incorporation of the amines was the same as that at which methylamine is incorporated in the absence of a proteinase. The nucleophile-reactive site revealed in alpha 2M after reaction with a proteinase was shown to decay with a t1/2 of 112 s, at pH 7.5. After the reaction with a proteinase or with methylamine, a free thiol group was detectable on each subunit of alpha 2M. We propose that the site for incorporation of methylamine in each subunit is a thiol ester, which in S-alpha 2M (the electrophoretically "slow" form) is sterically shielded from reaction with large nucleophiles, but is revealed as a highly reactive group, free from steric hindrance, after the proteolytic cleavage. We have designated the activated species of the molecule "alpha 2M".

Hepcidin Bound to α2-Macroglobulin Reduces Ferroportin-1 Expression and Enhances Its Activity at Reducing Serum Iron Levels

“…Activation of ␣ 2 M by Methylamine-Conversion of native ␣ 2 M into its activated form (␣ 2 M-MA) was achieved by standard methods using methylamine (14,19,20). Briefly, activation of ␣ 2 M was performed by incubation with 200 mM methylamine, 0.05 M Tris-HCl (pH 8.1) for 4 h at room temperature (28).…”

“…lanes 3 and 7). A potential explanation for this finding is that pre-existing hepcidin bound to native ␣ 2 M was released during the methylamine-triggered conformational change (19,20), which was subsequently removed by Sephadex G-25 filtration column. Both ␣ 2 M⅐hepcidin (lane 6) and ␣ 2 MMA⅐hepcidin (lane 8) were able to significantly (p Ͻ 0.001) reduce Fpn1 expression in J774 cells to a similar extent as hepcidin alone (Fig.…”

“…Notably, ␣ 2 M-MA closely resembles the structure and function of protease-activated ␣ 2 M, and hence, it is widely utilized to mimic the activated state of this protein (19,20). Activation of ␣ 2 M triggers a conformational change resulting in a shift of migration as resolved by native PAGE, with the fast band representing ␣ 2 M-MA and the slow band being native ␣ 2 M (Fig.…”

“…It is known that the activity of ␣ 2 M in inhibiting proteases is mediated via protease binding to a "bait region" of ␣ 2 M, which results in a conformational change to its active form (18), and this can be mimicked by methylamine treatment (14,19,20). Activated ␣ 2 M is rapidly internalized by endocytosis via binding to low density lipoprotein receptor-related protein-1 (Lrp1) (18).…”

Sequence determination of the thiolester site of the fourth component of human complement.

“…Nucleophile inactivation, autolytic cleavage on denaturation, and the ability to form covalent bonds (with proteases) have also been described for a2-macroglobulin (a2M) (25)(26)(27)(28)(29)(30)(31). We and others have shown that nucleophile inactivation again correlates with the appearance of a thiol group (10,(32)(33)(34). Thus, it appears that C3, C4, and a2M share a common reaction mechanism.…”