Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function.

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.

Familial hemophagocytic lymphohistiocytosis in an adult patient homozygous for A91V in the perforin gene, with tuberculosis infection.

Perforin gene (PRF1) mutations have been reported in 20-30% of patients with familial hemophagocytic lymphohistiocytosis (FHL), an autosomal recessive disorder of infancy and early childhood that impairs or abolishes lymphocyte cytotoxicity. We report the first case of FHL in an adult patient homozygous for A91V in PRF1 with tuberculosis. The monozygotic twin of the patient is healthy. A91V confers genetic susceptibility for the development of FHL, but is not enough to trigger the disease on its own. We discuss the role of the A91V change together with M. tuberculosis infection as synergistic factors in the late onset of FHL.