Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM 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 α2-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 histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. 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.

中文翻译：

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由于硫羟酸酯的反应性增强，α2-巨球蛋白样蛋白 1 可以通过其羟基结合并抑制蛋白酶。


α-巨球蛋白 (αM) 超家族中的蛋白质在蛋白水解激活后使用硫羟酸酯形成共价缀合产物。 αM 蛋白酶抑制剂使用它们来缀合蛋白酶并优先与伯胺（例如赖氨酸侧链）反应，而 αM 补体成分 C3 和 C4B 的羟基反应性增加，通过保守的组氨酸残基传递并允许缀合到细胞表面聚糖。人 α2-巨球蛋白样蛋白 1 (A2ML1) 是一种单体蛋白酶抑制剂，但具有传递组氨酸残基的羟基反应性。在这里，我们通过比较重组 WT A2ML1 和去除组氨酸的 A2ML1 H1084N 突变体，研究了羟基反应性在蛋白酶抑制剂中的作用。两种 A2ML1 的硫羟酸酯均与胺底物甘氨酸发生反应，但只有 WT A2ML1 与羟基底物甘油发生反应，表明 His-1084 增加了 A2ML1 的硫羟酸酯的羟基反应性。尽管 A2ML1 都缀合并抑制嗜热菌蛋白酶，但乙酰化嗜热菌蛋白酶需要 His-1084 来缀合和抑制，而乙酰化嗜热菌蛋白酶缺乏伯胺。使用 MS，我们鉴定了嗜热菌丝氨酸残基和 A2ML1 硫羟酸酯之间形成的酯键。这些结果表明，组氨酸增强的羟基反应性有助于 αM 蛋白对蛋白酶的抑制。 His-1084在pH 5时没有改善A2ML1的蛋白酶抑制作用，表明A2ML1的羟基反应性不适应其酸性表皮环境。