Fu Zhirong, Thorpe Michael, Alemayehu Rahel, Roy Ananya, Kervinen Jukka, de Garavilla Lawrence, Åbrink Magnus, Hellman Lars
Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, SE-751 24 Uppsala, Sweden.
Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden.
J Immunol. 2017 Feb 15;198(4):1474-1483. doi: 10.4049/jimmunol.1601223. Epub 2017 Jan 4.
Human mast cell chymase (HC) and human neutrophil cathepsin G (hCG) show relatively similar cleavage specificities: they both have chymotryptic activity but can also cleave efficiently after leucine. Their relatively broad specificity suggests that they may cleave almost any substrate if present in high enough concentrations or for a sufficiently long time. A number of potential substrates have been identified for these enzymes and, recently, these enzymes have also been implicated in regulating cytokine activity by cleaving numerous cytokines and chemokines. To obtain a better understanding of their selectivity for various potential in vivo substrates, we analyzed the cleavage of a panel of 51 active recombinant cytokines and chemokines. Surprisingly, our results showed a high selectivity of HC; only 4 of 51 of these proteins were substantially cleaved. hCG cleaved a few additional proteins, although this occurred after adding almost equimolar amounts of enzyme to target. The explanation for this wide difference in activity against peptides or other linear substrates compared with native proteins is most likely related to the reduced accessibility of the enzymes to potential cleavage sites in folded proteins. In this article, we present evidence that sites not exposed on the surface of the protein are not cleaved by the enzyme. Interestingly, both enzymes readily cleaved IL-18 and IL-33, two IL-1-related alarmins, as well as the cytokine IL-15, which is important for T cell and NK cell homeostasis. Cleavage of the alarmins by HC and hCG suggests a function in regulating excessive inflammation.
人肥大细胞糜酶（HC）和人中性粒细胞组织蛋白酶G（hCG）表现出相对相似的切割特异性：它们都具有胰凝乳蛋白酶活性，但也能在亮氨酸之后有效地切割。它们相对宽泛的特异性表明，如果浓度足够高或作用时间足够长，它们可能切割几乎任何底物。已经为这些酶鉴定了许多潜在底物，最近，这些酶还被认为通过切割多种细胞因子和趋化因子来调节细胞因子活性。为了更好地了解它们对各种潜在体内底物的选择性，我们分析了一组51种活性重组细胞因子和趋化因子的切割情况。令人惊讶的是，我们的结果显示HC具有高度选择性；这些蛋白质中只有4种被大量切割。hCG切割了一些其他蛋白质，尽管这是在向靶标添加几乎等摩尔量的酶之后才发生的。与天然蛋白质相比，这些酶对肽或其他线性底物的活性存在巨大差异，其原因很可能与酶对折叠蛋白质中潜在切割位点的可及性降低有关。在本文中，我们提供证据表明，蛋白质表面未暴露的位点不会被该酶切割。有趣的是，这两种酶都能轻易切割IL-18和IL-33这两种与IL-1相关的警报素，以及对T细胞和NK细胞稳态很重要的细胞因子IL-15。HC和hCG对警报素的切割表明其在调节过度炎症中发挥作用。
