The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region
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
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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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 12

Effect of MMP-7 on structure and cytotoxicity of human sFasL.

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Effect of MMP-7 on structure and cytotoxicity of human sFasL. (A) Incuba...
(A) Incubation of the long form of the rh-sFasL (0.5 μM) with serial concentrations of MMP-7 (0.16, 0.5, or 1.5 μM). The samples were analyzed by Western blot using SDS-PAGE in nonreducing conditions using a polyclonal antibody against human sFasL and (B) tested for cytotoxicity in Jurkat cells. (A) Treatment with serial concentrations of MMP-7 (lanes 2–4) resulted in a shift of the monomers (25 kDa) of sFasL to a lower molecular weight and the appearance of a new band below 10 kDa (arrow) corresponding to the predicted molecular weight of the stalk region plus the 15–amino acid linker peptide encoded by the expression vector. Numbers on the left show molecular weight. (B) Cytotoxicity of native and MMP-7–treated rh-sFasL in Jurkat cells at 18 hours using the alamarBlue assay. The white bar shows the cytotoxicity of untreated sFasL; the gray bars show the cytotoxicity of MMP-7 alone. Incubation with MMP-7 reduced the cytotoxicity of long human sFasL (black bars) as compared with the effect of MMP-7 alone (gray bars). The data are mean ± SD of 3 separate experiments performed in duplicate. *P < 0.05 vs. long human sFasL and MMP-7 alone.