Aberrant maturation of mutant perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis
Kimberly A. Risma, … , Alexandra H. Filipovich, Janos Sumegi
Kimberly A. Risma, … , Alexandra H. Filipovich, Janos Sumegi
Published January 4, 2006
Citation Information: J Clin Invest. 2006;116(1):182-192. https://doi.org/10.1172/JCI26217.
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Research Article Immunology

Aberrant maturation of mutant perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis

Abstract

Missense mutations in perforin, a critical effector of lymphocyte cytotoxicity, lead to a spectrum of diseases, from familial hemophagocytic lymphohistiocytosis to an increased risk of tumorigenesis. Understanding of the impact of mutations has been limited by an inability to express human perforin in vitro. We have shown, for the first time to our knowledge, that recombinant human perforin is expressed, processed appropriately, and functional in rat basophilic leukemia (RBL) cells following retroviral transduction. Subsequently, we have addressed how perforin missense mutations lead to absent perforin detection and impaired cytotoxicity by analyzing 21 missense mutations by flow cytometry, immunohistochemistry, and immunoblot. We identified perforin missense mutations with partial maturation (class 1), no apparent proteolytic maturation (class 2), and no recognizable forms of perforin (class 3). Class 1 mutations exhibit lytic function when expressed in RBL cells and are associated with residual protein detection and variable cytotoxic function in affected individuals, suggesting that carriers of class 1 alleles may exhibit more subtle immune defects. By contrast, class 3 mutations cause severely diminished perforin detection and cytotoxicity, while class 2 mutations have an intermediate phenotype. Thus, the pathologic mechanism of perforin missense mutation likely involves a protein dosage effect of the mature protein.

Authors

Kimberly A. Risma, Robert W. Frayer, Alexandra H. Filipovich, Janos Sumegi

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Figure 8

Western blot analysis of RBL-1 expressing PRF1 missense mutations with absent mature band formation.

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Western blot analysis of RBL-1 expressing PRF1 missense mutations with a...
(A) Mutant proteins demonstrated an absence of the mature band by nonreducing SDS-PAGE followed by Western blot using antibodies as labeled. WT perforin is shown for comparison. The use of 2 different antibodies (H315 and P1-8) was critical to demonstrate the presence of the 70-kDa band under reducing conditions for PRF1-F157V and PRF1-R225W. Fifty micrograms protein was loaded per lane. (B) RBL-1 cells expressing WT, V50M, and R225W perforin were disrupted and fractionated through Percoll gradients. Samples from every second fraction of the gradients were then analyzed for the presence of precursor and mature forms of perforin on nonreducing SDS-PAGE. WT perforin is shown for comparison. “C” indicates a cell lysate from RBL-1 cells expressing WT human perforin (4 μg protein). For PRF1-V50M, 4 μg protein was loaded per lane; 1.5 μg was loaded for WT PRF1 and PRF1-R225W. Detection was by P1-8 antibody.