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Biosynthesis of granulysin, a novel cytolytic molecule
D A Hanson1, A A Kaspar, F R Poulain
1Department of Pediatrics, Stanford University School of Medicine, CA 94305-5208, USA.
Abstract:
Granulysin is a newly described lytic molecule expressed by CTL and NK cells. Three mRNA (519, 520, and 522) and two protein products of 15 and 9 kDa are encoded by the granulysin gene. Stable transfectants overexpressing the predominate 520 mRNA were generated to determine the protein products originating from the translation of this message. A transfectant of the NK cell tumor YT overexpressed both 15 and 9 kDa proteins while a transfectant of the T cell tumor HuT78 produced mainly 15 kDa granulysin. Thus the 520 mRNA is sufficient for production of both 15 and 9 kDa granulysin. 9 kDa granulysin accumulated via post-translational processing of 15 kDa protein and was present intracellularly but not in the cell culture supernatant, indicating specific retention of the 9 kDa protein. An inhibitor of granule acidification, concanamycin A, blocked the processing of 15 kDa granulysin to the 9 kDa form. A deduced structural difference between the two forms of the protein and a decrease in lytic activity of 9 kDa granulysin at granule pH suggest two mechanisms by which a granulysin expressing cell is protected from autolysis during the biosynthesis of this potentially harmful molecule.
Insights
Granulysin, a cytotoxic molecule from CTL and NK cells, is produced in 15 and 9 kDa forms from the 520 mRNA. Processing of the 15 kDa form to 9 kDa is blocked by inhibiting granule acidification, protecting cells from self-damage.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Granulysin is a cytotoxic molecule expressed by cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells.
- The granulysin gene encodes three mRNA variants (519, 520, 522) and two protein products (15 kDa and 9 kDa).
Purpose of the Study:
- To investigate the protein products derived from the predominant 520 mRNA of granulysin.
- To elucidate the post-translational processing and cellular localization of granulysin protein forms.
- To understand the mechanisms protecting cells from granulysin-mediated autolysis.
Main Methods:
- Generation of stable transfectants overexpressing the 520 mRNA in YT (NK) and HuT78 (T) cell lines.
- Analysis of protein expression and processing using Western blotting.
- Investigation of the effect of granule acidification inhibitors (concanamycin A) on protein processing.
Main Results:
- The 520 mRNA is sufficient for producing both 15 kDa and 9 kDa granulysin proteins.
- YT cells produced both 15 and 9 kDa forms, while HuT78 cells primarily produced the 15 kDa form.
- 9 kDa granulysin results from post-translational processing of the 15 kDa form and is retained intracellularly.
- Inhibition of granule acidification blocked the conversion of 15 kDa to 9 kDa granulysin.
- Structural differences and reduced lytic activity of 9 kDa granulysin at acidic pH suggest autoprotective mechanisms.
Conclusions:
- The 520 mRNA variant of granulysin can generate both 15 kDa and 9 kDa protein products.
- Intracellular processing and retention of the 9 kDa form, influenced by granule pH, protect expressing cells from autolysis.
- Granulysin biosynthesis involves self-protection mechanisms to prevent damage to the producing cytotoxic cells.