A Triple-Mutated Allele of Granzyme B Incapable of Inducing Apoptosis
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A triple-mutated allele of granzyme B incapable of inducing apoptosis Dorian McIlroy*†, Pierre-Franc¸ois Cartron‡, Pierre Tuffery§, Yasmine Dudoit*, Assia Samri*, Brigitte Autran*, Franc¸ois M. Vallette‡, Patrice Debre´ *, and Ioannis Theodorou*¶ *Laboratoire d’Immunologie Cellulaire et Tissulaire, Institut National de la Sante´et de la Recherche Me´dicale U543, Faculte´deMe´ decine Pitie´-Salpeˆtrie`re, 75013 Paris, France; ‡Institut National de la Sante´et de la Recherche Me´dicale U419, Institut de Biologie, 44000 Nantes, France; and §Institut National de la Sante´et de la Recherche Me´dicale U436, Equipe de Bioinformatique Ge´nomique et Mole´culaire, Universite´de Paris 7, 75013 Paris, France Communicated by Jean Dausset, Fondation Jean Dausset–Centre d’E´ tude du Polymorphisme Humain, Paris, France, December 27, 2002 (received for review June 6, 2002) Granzyme B (GzmB) is a serine protease involved in many pathol- resistant cell lines (20, 21). However, no congenital human ogies, including viral infections, autoimmunity, transplant rejec- diseases have been linked to the disruption of the GzmB gene, tion, and antitumor immunity. To measure the extent of genetic and GzmB knockout mice have no developmental or hemato- variation in GzmB, we screened the GzmB gene for polymorphisms logical abnormalities (22). Therefore, we hypothesized that, in and defined a frequently represented triple-mutated GzmB allele. contrast to mutations in the perforin gene (23), coding poly- 48 88 245 In this variant, three amino acids of the mature protein Q P Y morphisms in the GzmB gene might be relatively well tolerated ؉ 245 88 48 are mutated to R A H .InCD8 cytotoxic T lymphocytes, GzmB in humans and, if present, could influence the killing of tumor ͞ was expressed at similar levels in QPY homozygous, QPY RAH cells. heterozygous, and RAH homozygous individuals, demonstrating To investigate this hypothesis, we screened the GzmB gene for that RAH GzmB is a stable protein. Active RAH GzmB expressed in single-nucleotide polymorphisms (SNPs) and thereby identified glioblastoma cell lines displayed proteolytic activity, but in contrast a common GzmB allele coding for a triple-mutated protein, the to QPY GzmB, it did not accumulate in the nucleus and was unable structure of which was investigated by molecular modeling. The to induce Bid cleavage, cytochrome c release, or apoptosis. Molec- expression of GzmB and the cytolytic activity in CTL from ular modeling showed that the three amino acid substitutions clustered near the C-terminal ␣-helix of the protein, indicating that individuals homozygous and heterozygous for the variant allele this region of the protein may be involved in the intracellular was investigated. The enzymatic activity and proapoptotic func- targeting of GzmB. The triple-mutated GzmB allele that we de- tions of variant GzmB were tested in transfected glioblastoma scribe appears to be incapable of inducing apoptosis in tumor cell lines, which revealed striking functional differences between the lines, and its presence could, therefore, influence both the prog- two alleles. nosis of cancer patients and the success rates of antitumor cellular Materials and Methods immunotherapy. Patient Samples. HIVϩ patient DNA samples were obtained from the French Immunoco cohort, and control DNA samples were ranzyme B (GzmB) is a protease of the chymotrypsin family from healthy blood donors. DNA samples from West African Gexpressed by differentiated cytotoxic T lymphocytes (CTL; ref. 1), as well as certain other cell types, such as activated and Vietnamese individuals were from HIV seropositive and keratinocytes (2). In cytotoxic cells, GzmB is a component of seronegative donors. All DNA samples were obtained from cytolytic granules (3). It is released upon target cell recognition, individuals enrolled in clinical studies approved by the relevant then specifically endocytosed by target cells via the cation local or national ethical review board. DNA was extracted from independent mannose 6-phosphate receptor (4). In the presence peripheral blood mononuclear cell (PBMC) by using QIAamp of perforin (5), which is also released from cytolytic granules (6), mini extraction kits (Qiagen, Valencia, CA). GzmB escapes from the endolysosomal compartment and gains Resected tumor tissue was obtained from adult glioblastoma access to a number of key proteins involved in the execution of patients undergoing surgery at the Department of Neurosurgery, the apoptotic program. Laennec Hospital (Nantes, France). Primary glioblastoma lines GzmB has a substrate specificity unique among serine pro- were derived from tumor samples as described (24). teases. It cleaves after aspartate residues, and experiments combining synthetic peptide and phage libraries have defined the PCR and Mutation Detection. A 4.0-kb PCR product encompassing extended specificity of GzmB as IEPD͞xG (7). This hexapeptide exons 1–5 of the GzmB gene was amplified using the ELT PCR is remarkably similar to the optimal substrate for caspase-8, an kit (Roche Molecular Biochemicals). This PCR product was then initiator caspase at the apex of the apoptotic signaling cascade used as a template for amplification of 200– to 450-bp segments activated by the ligation of the Fas death receptor, and this encompassing each exon of the gene. PCR products were similarity explains the proapoptotic properties of GzmB. In dehybridized͞rehybridized, and heteroduplex formation was de- particular, cleavage of Bid by GzmB activates the mitochondrial tected by denaturing HPLC (dHPLC) by using a Transgenomic pathway of programmed cell death (8, 9), cleavage of caspase-3 Wave instrument. Mutations were identified by sequencing of directly generates cytoplasmic caspase activity (10), and caspase- PCR products from heterozygous individuals. Linkage disequi- activated DNase (CAD) is released by GzmB cleavage of the inhibitor protein, ICAD, leading to nuclear DNA fragmentation (11, 12). Abbreviations: GzmB, granzyme B; CTL, cytotoxic T lymphocyte; CAD, caspase-activated Like other cytotoxic granule contents, GzmB is involved in DNase; SNP, single-nucleotide polymorphism; PBMC, peripheral blood mononuclear cell; several pathologies including viral infections (13), graft rejec- ALT, long-term asymptomatic. tion, graft-vs.-host disease (14–17), and rheumatoid arthritis (18, †Present address: Institut National de la Sante´et de la Recherche Me´dicale U419, Institut de Biologie, 9 Quai Moncousu, 44000 Nantes, France. 19). GzmB is also likely to play an important role in antitumor ͞ ¶To whom correspondence should be addressed at: Laboratoire d’Immunologie Cellulaire immune responses. The perforin GzmB pathway efficiently et Tissulaire, Institut National de la Sante´ et de la Recherche Me´dicale U543, Baˆtiment induces tumor cell death in vitro and is also able to induce CERVI, CH Pitie´-Salpeˆtrie`re, 83 Boulevard de l’Hoˆpital, 75013 Paris, France. E-mail: apoptosis in multiple-drug-resistant and death-receptor- [email protected]. 2562–2567 ͉ PNAS ͉ March 4, 2003 ͉ vol. 100 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0437935100 Downloaded by guest on September 30, 2021 librium was confirmed by sequencing of long PCR products a semidry transfer system (Bio-Rad). Membranes were blocked cloned into the TA cloning vector (Invitrogen). in PBS 0.05% Tween 20 containing 10% (vol͞vol) skim milk Individuals heterozygous for SNPs in exon 2 (48Q ϭϾR) and and then incubated with the following mouse monoclonal anti- exon 3 (88P ϭϾA) and exon 5 (245Y ϭϾH) of the gene were bodies: anti-human GzmB (clone 2C5, Santa Cruz Biotechnol- typed by dHPLC. Individuals homozygous for mutations in these ogy); anti-actin (Chemicon); anti-cytochrome c (R & D Sys- exons were identified by hybridizing PCR products with a tems); anti-cytochrome oxidase subunit IV (Molecular Probes), standard wild-type PCR product and then typed by dHPLC. The anti-ICAD͞DFF-45 (Santa Cruz Biotechnology), anti-caspase-3 C3057A SNP in intron 3 was detected by NgoM IV restriction (PharMingen), anti-caspase-8 (PharMingen) or a polyclonal fragment-length polymorphism (RFLP). rabbit antibody against human Bid (kindly provided by J. C. Martinou, University of Geneva, Geneva). Bands were visual- Flow Cytometry. PBMCs were thawed and then stained for ized by chemiluminescence (Amersham Pharmacia) after incu- surface CD8 and intracellular GzmB either immediately or after bation with the appropriate peroxidase-conjugated secondary 4 days culture in RPMI medium 1640 containing 10% (vol͞vol) antibodies. FCS (Invitrogen), 1% (vol͞vol) MEM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 2 mM L- Cell Death and GzmB Activity Assays. Cell death was quantified by glutamine (Invitrogen), 100 units͞ml penicillin, 100 g͞ml strep- using the Cytotox 96 NonRadioactive Cytotoxicity Assay kit tomycin, 0.25 g͞ml amphotericin B, and 1 g͞ml PHA-P (Promega). Caspase 3 (DEVDase) activity in Nonidet P-40 (Sigma). Cells were surface-labeled with anti-CD8 APC lysates was measured by using the Caspace Assay kit (Promega) (PharMingen) and anti-CD56 PE-Cy5 (Beckman Coulter), containing the fluorescent substrate DEVD-7-amino-4-methyl washed, and then permeabilized by using the Fix and Perm kit coumarin (AMC). GzmB activity in cell lysates was measured by (Caltag, South San Francisco, CA). During permeabilization, monitoring cleavage of IETD-AMC. In some experiments, cells were incubated with 10 g͞ml anti-GzmB PE (clone GB12, caspase activity was blocked by the addition