The Functional Basis for Hemophagocytic Lymphohistiocytosis in a Patient with Co-inherited Missense Mutations in the Perforin (PFN1) Gene

Abstract

About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated ( ‫ف‬ 45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure-function relationships for lymphocyte perforin.

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

“…Before recombinant Gzm/Pfp treatment, cells were washed three times in DMEM media containing 0.5% BSA (Fraction V, Roche Diagnostics GmbH, Basel, Switzerland). Recombinant human GzmB, hGzmA or mGzmA were diluted in DMEM/BSA buffer and added to target cells before addition of a sublytic dose of recombinant mouse Pfp (purified in house to a concentration of 300 mg/ml, as previously described 41 ).…”

Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer

“…Western immunoblotting was performed using reducing or nonreducing Laemmli SDS/PAGE loading buffer, and PRF was detected using rat anti-mouse mAb (P1-8, provided by H. Yagita, Juntendo University, Tokyo) as described in ref. 29. Following transfection, the cells were cultured at 37°C in 5% CO 2 (mouse lymphocytes) or 10% CO 2 (RBL-2H3 cells) for 18 -24 h. To test for temperature sensitivity, cells were cultured at 30°C for 18 -24 h after transfection.…”

“…Primary CTL culture, transfection using the Amaxa Nucleofector Technology, FACS sorting of transfected lymphocytes, and 51 Cr release cytotoxicity assays were all performed as described (19). The culture of rat basophil leukemia (RBL)-2H3 cells, gene transfection, FACS sorting, and cytotoxicity assays were also carried out as previously described (29). Western immunoblotting was performed using reducing or nonreducing Laemmli SDS/PAGE loading buffer, and PRF was detected using rat anti-mouse mAb (P1-8, provided by H. Yagita, Juntendo University, Tokyo) as described in ref.…”

Granzyme K cleaves the nucleosome assembly protein SET to induce single-stranded DNA nicks of target cells

“…Loading GzmK with perforin was performed as described previously. 15,40 Briefly, a sublytic dose was first titrated with recombinant perforin. Then the sublytic dose of perforin was used in combination with GzmK or S-AGzmK at each assay.…”