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α2-Macroglobulin-like protein 1 can conjugate and inhibitproteases through their hydroxyl groups, because of anenhanced reactivity of its thiol ester

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Proteins in the a-macroglobulin (aM) superfamily use thiol esters to formcovalent conjugation products upon their proteolytic activation. aM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of aM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α 2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidineenhanced hydroxyl reactivity can contribute to protease inhibition by anaMprotein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Original languageEnglish
JournalJournal of Biological Chemistry
Pages (from-to)16732-16742
Number of pages11
Publication statusPublished - Dec 2020

Bibliographical note

Publisher Copyright:
© 2020 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.

Copyright 2020 Elsevier B.V., All rights reserved.

    Research areas

  • &#945, 2-macroglobulin, A2ML1, ALPHA-MACROGLOBULIN, COMPLEX, COVALENT-BINDING, HUMAN ALPHA-2-MACROGLOBULIN, INSIGHTS, INTERNAL THIOESTER, LOCALIZATION, MECHANISM, SEQUENCE, SYSTEM, alpha 2 macroglobulin like protein 1, alpha-2-macroglobulin, complement, inhibition mechanism, mutagenesis, mutagenesis in vitro, protease, protease inhibitor, protein crosslinking, thiol ester

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