Cloning, Expression And Apoptosis Inducing Activity Of Perforin

Perforin plays an essential role in the granule exocytosis pathway of target-cell apoptosis, the main way cytotoxic T cells (CTL) and natural killer (NK) cells eliminate virus-infected cells and tumors. Apoptotic target-cell death is triggered by release of cytotoxic granule contents into the immunological synapse formed by CTL binding to its target cell. Cytotoxic granules contain perforin and a group of serine proteases called granzymes in a proteoglycan matrix. Perforin delivers granzymes into the target-cell cytosol. Because multiple granzymes can independently trigger target-cell apoptosis, targeted disruption of any one granzyme gene in mice only modestly affects immune protection. However, there is only one known delivery molecule. Consequently, mice deficient in perforin are profoundly immunodeficient and are unable to protect themselves against viral infection and tumors. Humans with familial hemophagocytic lymphohistiocytosis due to perforin gene mutations also have compromised antiviral immunity. Although perforin was identified almost 20 years ago and cloned shortly thereafter, the molecular and cellular basis for perforin activity remains uncertain.Perforin shares homology with terminal complement components. Because, like complement, perforin multimerizes in membranes to form pores, perforin was originally hypothesized to deliver granzymes to target cells through plasma membrane pores. However, perforin pores seen on electron micrographs may be too small (≤50 nm diameter) to act as channels for globular molecules like granzymes. The plasma membrane-pore model was questioned when granzyme B was shown to be endocytosed without perforin. How extracellular perforin acting on the plasma membrane could target granzyme release from membrane-bound intracellular vesicles was still unclear. The latest work declared that perforin creates pores in the target-cell membrane during cell-mediated lysis, transiently allowing Ca2+ into the cell. The Ca2+ flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Meanwhile, the pores are also subjected to clearance by endocytosis, during which perforin may exert certain endosomolytic function and release granzymes and/or itself into cytosol. It has already been clearly demonstrated that the translocated cytosol granzymes could interact with their substrates to induce apoptotic cell death; however, people are still unsure whether the perforin molecule would stop its work at this stage.In order to understand the function of perforin after the endocytosis induced by target cells'wounded membrane-repair response, or other possible roles this molecule plays in target-cell killing, allotropic expression strategy was used on insentive studies of the human perforin gene (PRF1). The full-length PRF1 cDNA was obtained through RT-PCR from the IL-2-stimulated human T cell leukemia derived cell line Jurkat. And, the sequence was subcloned into pcDNA3.1-A vector to generate pcDNA3.1-A/PRF1. The full-length perforin are predicted to be biosynthesized, modified and then stored in cytotoxic lymphocytes'granules. However, no one knows how it would work if perforin was produced by other cell types. On the other hand, to disclose the potency of cytosol localized perforin, we also construct a truncated perforin in which the signal peptide was removed from N-terminus of PRF1 cDNA and the conserved C2 domain was reserved as the C-terminal sequence according to the NCBI database(genbank,Accession#:NP₀05032.1;GI:4826942). As we known, N-linked glycosylation site in the C-terminal of perforin would be modified during its biosynthesis and the bulky glycan modification may shield its calcium-dependent phospholipids-binding C2 domain to inhibit its cytotoxity. Deletion of the C-terminal regions beside the C2 is predicted to directly create an active perforin form, whereas the absence of a signal peptide may cause sequestration of the active perforin inside the cytosol after it was initially generated. In short, we could also call it interior expressed active form. This truncated PRF1 was also subcloned into pcDNA3.1-A vector and named as pcDNA3.1-A/PRF1-t1. Besides, LacZ insertion (pcDNA3.1/lacZ) as well as blank vector (pcDNA3.1-A) are taken as controls.Growth inhibition of both HepG2 and SK-BR-3 cells was detected by the cells counting assay after either form of our recombinant PRF1 was transiently transfected. Meanwhile, PRF1 expression in SK-BR-3 cells was confirmed by RT-PCR and indirect immunofluorescent staining. Interestingly, although the viability of HeLa cells was also greatly impaired (similar with HepG2 and SK-BR-3), both forms of perforin didn't affect the living status of Jurkat cells, from which PRF1 was cloned. Such a T cell leukemia cell line may own certain unknown protective mechanisms to make itself insensitive to perforin expression.To define what really happened in PRF1 transfected cells, morphologic features of HepG2 cell was detected by electron microscopy. Nuclei shrinkage, DNA condensing and apoptotic body which characterize apoptotic cell death, were clearly observed 32 h after both PRF1s transfection whereas membrane disruption and low electron density in cytoplasm were exceptionally increased in pcDNA3.1-A/PRF1 transfectants. Similar results were congruously obtained when Annexin-V/PI dual stain was adopted on the same cell line or other cell lines such as SK-BR-3 and HEK293. The Annexin-V+PI- cells portion, which typify early apoptosis, were all increased after treatment with both recombinant PRF1 compared with controls. If necrotic cell death (PI+) was simultaneously considered, the pcDNA3.1-A/PRF1 transfection exhibited more potent cytotoxicity as reflected by a higher PI positive cells portion, whereas pcDNA3.1-A/PRF1-t1, which mainly induced apoptotic cell death, just contribute less to overall cell death. Moreover, the apoptotic cell death induced by both full-length and truncated perforin was also verified by TUNEL assay in SK-BR-3 and HepG2 cells 48 h after transfection and Caspase-3 activation assay in HEK293 cells 36 h after transfection.Former studies emphasized that perforin was mainly involved in granzymes delivery, and granzymes were exclusively determinant apoptosis executioner. However, in absence of granzymes in our allotropic expression system, perforin seemed to induce apoptotic cell death by itself. To examine the pathway involved in this process, key molecules of mitochondria apoptotic proteins (e.g., cytochrome C and AIF) were detected by indirect immunofluorescent staining of SK-BR-3 cells 36 h after transfection. Morphologic features under the fluorescence microscope declared that DNA condensing and nuclei distortion or disruption reflected by DAPI stain were always accompanied by allotropic expression of either of the perforin forms, among which cytosol or nuclei translocations of mitochondria cytochrome C or AIF were notablely increased. On the other hand, if inhibitors (pan caspases inhibitor Z-VAD-FMK) or protectors (mitochondria membrane potential stabilizer Bcl-XL BH44-23) were adopted before transfection, AIF translocations, nuclei damages as well as overall cell death were all markedly suppressed. Therefore, allotopic-expressed perforin seemed to interfere with some subcellular organelles (e.g., mitochondria) to induce apoptotic cell death.Although the allotropic expression strategy revealed a potential apoptosis-inducing activity of perforin, it may not faithfully replicate what happens when a killer cell destroys a target cell and where perforin and/or granzymes are delivered or co-delivered in the spatially limited region of the immunological synapse. To determine the subcellular function of perforin in cells killing process above, an immunological synapse mediated effector-target cells killing model was needed.As our former experiments implied that Jurkat cells could protect themselves during recombinant perforin allotropic expression, full-length PRF1 was transiently transfected in Jurkat cells for 32 h and then co-cultured through transwell with SK-BR-3 cells spread on slides before to detect the trace of allotropic expressed PRF1. To our surprise, instead of stored in cytosol granule, perforin was directly secreted and the SK-BR-3 cells on the slides could be labeled by perforin antibody through indirect immunofluorescent staining after co-cultured for 72 h. Furthermore, perforin mRNA was detected by RT-PCR in full-length PRF1 stably transfected Jurkat cell clones, and the secreted recombinant protein was detected in cultured surpernants. Such a Jurkat cell clone is also proved to impaire the viability of co-cultured SK-BR-3 cells.On the other hand, Based on one of receptors/ligands (CD226/CD155, CD112) interaction which was indispensable in immunological synapse formation and was proved to enhance the NK-mediated lysis of tumor cells, we tried to build the effector-target cells killing model by the possible linkage between the Jurkat cells stable clone in which CD155 and CD112 are constitutively expressed, and a CHO cells clone in which the recombinant CD226 is stably expressed on cell surface. However, no obvious cells interaction or killing was detected when those cells are mix-cultured.In summary, full-length and an interior expressed active (truncated) form of PRF1 was cloned from Jurkat cells, and allotropic expression either of these perforin forms could induce apoptotic cell death, though the full-length is also necrotizing. The apoptosis may be induced by the destruction of some subcellular organelles (e.g., mitochondria), and could be partially suppressed by either pan caspases inhibitor Z-VAD-FMK or mitochondria membrane potential stabilizer Bcl-XL BH44-23. The allotropic expression strategy implies the potential apoptosis-inducing ability of perforin which may benefit immune surveillance and defense, and might also be adopted for therapeutic purpose. Moreover, we are still continually struggling in building an immunological synapse-mediated effector-target cells killing model to verify our findings.The present study provides new insight into the critical role of perforin in immune competent cells-mediated target-cell killing and a precise mechanism underlying perforin-induced apoptosis.