Spatially Resolved Monitoring of Neutrophil Elastase Activity with Ratiometric Fluorescent Reporters
Spatially Resolved Monitoring of Neutrophil Elastase Activity with Ratiometric Fluorescent Reporters
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DOI：
10.1002/anie.201109226
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Schultz, Carsten
中科院分区：
文献类型：
--
作者：
Gehrig, Stefanie;Mall, Marcus A.;Schultz, Carsten
Neutrophil elastase (NE), a serine protease mainly secreted by neutrophils under physiological and pathophysiological conditions, has important protective functions including remodeling of the extracellular matrix and host defense against bacterial infections.[1, 2] However, in diseases characterized by chronic neutrophilic inflammation such as cigarette-smoke-induced chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) lung disease, excessive NE activity has been implicated in proteolytic damage of the airways and lung parenchyma causing bronchiectasis and emphysema, ultimately leading to respiratory failure and death.[3–7] Indeed, mice that lack NE are partially resistant to cigarette-smoke-induced lung damage.[3] A critical role of the protease/antiprotease balance is also highlighted in patients who lack the major endogenous inhibitor of NE, α1-antitrypsin (A1AT); they develop early-onset emphysema,[8] indicating that proper regulation of NE activity is of major significance for global lung homeostasis. In neutrophils NE is stored in its active form in azurophilic granules and released upon stimulation.[1] Since local enzyme activity in the immediate vicinity of the cell membrane is required for migration of neutrophils to sites of inflammation, it was hypothesized that membrane-associated NE might be responsible for tissue transmigration, whereas released NE is inhibited in the airway lumen to protect the lung from uncontrolled proteolytic damage.[6] In this context, it appears to be crucial to understand the activity distribution and function of both cell-surface-associated NE and soluble NE. So far, studies on the role of NE in chronic lung disease have focused on the soluble form of NE,[4, 5, 9] generally measured using the chromophoric substrate NMeOSuc-AAPV-pNA or its fluorescent variant, which in humans displays good selectivity over the structurally related proteinase 3 (PR3).[10, 11] However, this specificity is mostly lost for the mouse variants of the enzymes.[12] Of note, the detection limit of assays using this substrate is in the nanomolar range, so that NE activity in biological samples such as bronchoalveolar lavage (BAL) is often too dilute to be detected.[4] Moreover, several reports demonstrated the presence of a membrane-bound fraction of NE,[13, 14] likely based on electrostatic interactions with the positively charged enzyme surface,[15] and the relative roles of membrane-associated versus soluble NE activity have to our knowledge not been studied.For reliable monitoring of protease activity in a complex matrix at the level of intact cells and tissues from mouse models and patient specimens, high specificity and sensitivity of the detection probe are mandatory. We therefore selected a longer peptide sequence fitting the modeled binding pockets of mouse and human NE in both directions from the substrate cleavage site.[16] The primary goal was the improved detection of mouse NE activity to permit the use of mouse models. As was previously successfully applied for the monitoring of matrix metalloproteinase 12 (MMP12) activity on cell membranes,[17] the new NE reporter was also lipidated. Measurements on cell surfaces not only provide local activity information, but also the signal accumulates and therefore a much lower reporter concentration is required, which is important for future clinical applications. We synthesized two specific small-molecule ratiometric NE monitoring reporters based on energy transfer: NEmo-1 to detect activity of the soluble enzyme and the lipidated variant NEmo-2 for insertion into the plasma membrane. We chose the peptide substrate sequence QPMAVVQSVPQ with a specific cleavage site …
