Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function
| Autor: | Michael B. Jordan, Ramon Urrea Moreno, Elena Cela, Juana Gil, Victor LaFay, Carmen Rodríguez-Sainz, Brian Oloizia, Andrew B. Herr, Janos Sumegi, Kimberly A. Risma |
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| Rok vydání: | 2008 |
| Předmět: |
Immunology
Mutant Mutation Missense chemical and pharmacologic phenomena medicine.disease_cause Biochemistry Membrane Lipids Mice Cell Line Tumor medicine Cytotoxic T cell Animals Homeostasis Humans Cytotoxicity C2 domain Immunobiology MACPF Mutation Immunity Cellular biology Perforin Perforin Deficiency Cell Membrane hemic and immune systems Cell Biology Hematology Cell biology Protein Structure Tertiary Rats Amino Acid Substitution biology.protein Calcium T-Lymphocytes Cytotoxic |
| Zdroj: | Blood. 113(2) |
| ISSN: | 1528-0020 |
| Popis: | Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function. |
| Databáze: | OpenAIRE |
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