The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.

We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using si...

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Autores principales:	Osamu Kajikawa, Raquel Herrero, Yu-Hua Chow, Chi F Hung, Gustavo Matute-Bello
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d75d1137431f4693ae1775bd8ce25f97
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spelling	oai:doaj.org-article:d75d1137431f4693ae1775bd8ce25f972021-12-02T20:10:29ZThe bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.1932-620310.1371/journal.pone.0253260https://doaj.org/article/d75d1137431f4693ae1775bd8ce25f972021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0253260https://doaj.org/toc/1932-6203We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.Osamu KajikawaRaquel HerreroYu-Hua ChowChi F HungGustavo Matute-BelloPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 6, p e0253260 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Osamu Kajikawa Raquel Herrero Yu-Hua Chow Chi F Hung Gustavo Matute-Bello The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
description	We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.
format	article
author	Osamu Kajikawa Raquel Herrero Yu-Hua Chow Chi F Hung Gustavo Matute-Bello
author_facet	Osamu Kajikawa Raquel Herrero Yu-Hua Chow Chi F Hung Gustavo Matute-Bello
author_sort	Osamu Kajikawa
title	The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
title_short	The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
title_full	The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
title_fullStr	The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
title_full_unstemmed	The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
title_sort	bioactivity of soluble fas ligand is modulated by key amino acids of its stalk region.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/d75d1137431f4693ae1775bd8ce25f97
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The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.

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