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Blood, 8 January 2009, Vol. 113, No. 2, pp. 338-346. Prepublished online as a Blood First Edition Paper on October 16, 2008; DOI 10.1182/blood-2008-08-172924. This Article Full Text (PDF) Supplemental Table and Figures All Versions of this Article: blood-2008-08-172924v1 113/2/338    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Urrea Moreno, R. Articles by Risma, K. A Search for Related Content PubMed PubMed Citation Articles by Urrea Moreno, R. Articles by Risma, K. A Social Bookmarking                   What's this?  Previous Article  |  Next Article  Submitted August 7, 2008 Accepted September 21, 2008 Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function Ramon Urrea Moreno, Juana Gil, Carmen Rodriguez-Sainz, Elena Cela, Victor LaFay, Brian Oloizia, Andrew B. Herr, Janos Sumegi, Michael B. Jordan, and Kimberly A Risma* Division of Immunology, Hospital General Universitario Gregorio Maranon, Universidad Complutense de Madrid, Madrid, Spain Division of Pediatric Oncology, Hospital General Universitario Gregorio Maranon, Universidad Complutense de Madrid, Madrid, Spain Division of Allergy/Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, United States Division of Hematology/Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States * Corresponding author; email: kimberly.risma{at}cchmc.org. 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 two 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 following 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. Utilizing novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only non-mutated and Y438C-substituted perforin were capable of calcium-dependent, lipid binding. Additionally, 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. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

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