The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region
Raquel Herrero, … , Xiaoyun Fu, Thomas R. Martin
Raquel Herrero, … , Xiaoyun Fu, Thomas R. Martin
Published February 1, 2011
Citation Information: J Clin Invest. 2011;121(3):1174-1190. https://doi.org/10.1172/JCI43004.
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Research Article

The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region

Abstract

Acute lung injury (ALI) is a life-threatening condition in critically ill patients. Injury to the alveolar epithelium is a critical event in ALI, and accumulating evidence suggests that it is linked to proapoptotic Fas/FasL signals. Active soluble FasL (sFasL) is detectable in the bronchoalveolar lavage (BAL) fluid of patients with ALI, but the mechanisms controlling its bioactivity are unclear. We therefore investigated how the structure of sFasL influences cellular activation in human and mouse lungs and the role of oxidants and proteases in modifying sFasL activity. The sFasL in BAL fluid from patients with ALI was bioactive and present in high molecular weight multimers and aggregates. Oxidants generated from neutrophil myeloperoxidase in BAL fluid promoted aggregation of sFasL in vitro and in vivo. Oxidation increased the biological activity of sFasL at low concentrations but degraded sFasL at high concentrations. The amino-terminal extracellular stalk region of human sFasL was required to induce lung injury in mice, and proteolytic cleavage of the stalk region by MMP-7 reduced the bioactivity of sFasL in human cells in vitro. The sFasL recovered from the lungs of patients with ALI contained both oxidized methionine residues and the stalk region. These data provide what we believe to be new insights into the structural determinants of sFasL bioactivity in the lungs of patients with ALI.

Authors

Raquel Herrero, Osamu Kajikawa, Gustavo Matute-Bello, Yi Wang, Naoki Hagimoto, Steve Mongovin, Venus Wong, David R. Park, Nathan Brot, Jay W. Heinecke, Henry Rosen, Richard B. Goodman, Xiaoyun Fu, Thomas R. Martin

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Figure 5

Methionine residues are involved in oxidative multimerization of sFasL.

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Methionine residues are involved in oxidative multimerization of sFasL. ...
(A) Amino acid sequence of mouse native-sFasL and met-sFasL. Native sFasL contains 2 methionine residues (underlined), whereas met-sFasL contains 7 methionine residues (underlined) in the same locations as those in human sFasL. (B) Western blot analysis of the native form of mouse sFasL (native-sFasL) and a mutated form of mouse sFasL containing methionines at the location of methionine in human sFasL (mouse met-sFasL). Native sFasL (lanes 2–4) and met-sFasL (lanes 6–8) were incubated with increasing concentrations of HOCl (μM), and the reaction was stopped with l-methionine (2.5 nM). For comparison, l-methionine was added to the reaction before incubation with mouse native sFasL (lane 1) or met-sFasL (lane 5) to prevent protein oxidation. Nonoxidized mouse native sFasL and met-sFasL showed single bands at 25 kDa, corresponding to sFasL monomers (lanes 1 and 5). When oxidized, met-sFasL (lanes 6–8) multimerized more readily than native-sFasL (lanes 2–4). (C) Biological activity of normal and oxidized forms of mouse native sFasL and met-sFasL for mouse lung epithelial cells (MLE-15). Either media alone or rh-sFasL were used as controls. Bars show the mean ± SD of 3 separate experiments. *P < 0.05 compared with all other samples. The Western blot was run in SDS-PAGE in reducing conditions (2-ME and heat).