A triple-mutated allele of granzyme B incapable of inducing apoptosis

Abstract

Granzyme B (GzmB) is a serine protease involved in many pathologies, including viral infections, autoimmunity, transplant rejection, and antitumor immunity. To measure the extent of genetic variation in GzmB, we screened the GzmB gene for polymorphisms and defined a frequently represented triple-mutated GzmB allele. In this variant, three amino acids of the mature protein Q(48)P(88)Y(245) are mutated to R(48)A(88)H(245). In CD8(+) cytotoxic T lymphocytes, GzmB was expressed at similar levels in QPY homozygous, QPY/RAH heterozygous, and RAH homozygous individuals, demonstrating that RAH GzmB is a stable protein. Active RAH GzmB expressed in glioblastoma cell lines displayed proteolytic activity, but in contrast to QPY GzmB, it did not accumulate in the nucleus and was unable to induce Bid cleavage, cytochrome c release, or apoptosis. Molecular modeling showed that the three amino acid substitutions clustered near the C-terminal alpha-helix of the protein, indicating that this region of the protein may be involved in the intracellular targeting of GzmB. The triple-mutated GzmB allele that we describe appears to be incapable of inducing apoptosis in tumor cell lines, and its presence could, therefore, influence both the prognosis of cancer patients and the success rates of antitumor cellular immunotherapy.

Figure 1

SNPs in the coding region of the GzmB gene. Nucleotide positions are numbered according to GenBank accession no. M28879. Amino acid positions are numbered according to the bovine chymotrypsinogen A sequence. Nucleotide positions of the SNPs located in exons are as follows: 2364A>G (48Q>R); 2893A>G (74K>K); 2933C>G (88P>A); 2974C>T (101N>N); 4243T>C (245Y>H). (Inset) *, The presence of multiple substitutions in intron 3.

Figure 2

Expression of GzmB and CTL activity by GzmB genotype. PBMC from untreated long-term asymptomatic HIV⁺ patients enrolled in the ALT cohort were thawed, and GzmB expression in CD8⁺ lymphocytes was measured by intracellular fluorescence-activated cell sorter staining either directly (A and B) or after 4 days culture in the presence of 1 μg/ml PHA-P (C and D). Results are shown as the percentage of CD8⁺ lymphocytes staining positive for GzmB (A and C), and as the number of PE molecules bound per cell in the GzmB-positive population (B and D). Each symbol represents one patient. Anti-HIV CTL activity in cell lines derived from PBMC of HIV⁺ patients enrolled in the ALT cohort was tested against autologous EBV-immortalized B-lymphoma lines infected with recombinant vaccinia viruses expressing HIV gag, pol, env, nef, rev, tat, or vif proteins. For each HIV protein, the results of a ⁵¹Cr release assay were noted as positive or negative, and the number of positive results were scored for each patient (E). Each symbol represents one patient. GzmB genotypes are as follows: 0, QPY/QPY; 1, QPY/RAH; 2, RAH/RAH.

Figure 3

Proteolytic activity and apoptosis induction by QPY and RAH GzmB. Three independently derived glioblastoma lines (numbered 12, 38, and 48) were transfected with plasmids coding for QPY or RAH GzmB, or mock- transfected with pCDNA3, as indicated. (A) IETDase activity measured in crude lysates of transfected cells in the presence of 1 mM iodoacetamide. (B) DEVDase activity in lysates of transfected cells. (C) LDH activity in supernatants of transfected cells. Enzyme activities are expressed as fluorescence units per hour per microgram of protein. NS, no significant difference in enzymatic activity. **, P < 0.005 (Student's unpaired t test). (D) Western blot detection of GzmB in cells transfected with QPY and RAH GzmB, but not in mock-transfected cells. Bands in this blot were quantified three times. Actin/GzmB ratios were 0.87 ± 0.04 and 0.83 ± 0.04 for QPY and RAH GzmB, respectively. (E) Cleavage of ICAD-L, Bid, caspase-3, and caspase-8 and release of cytochrome c from mitochondria were detected only in cells transfected with QPY GzmB.

Figure 4

Subcellular localization of QPY and RAH GzmB. Primary glioblastoma cells were transfected with QPY (A–C and G–I) or RAH (D–F and J–L) GzmB, cultured for 14–18 h in the presence (G–L) or absence (A–F) of 1 μM staurosporine, then stained for GzmB (A, D, G, and J) and nuclear DNA (B, E, H, and K). Merged images are shown in C, F, I, and L.

Figure 5

Structure of QPY and RAH GzmB. (A and B) Representations of the complete QPY (A) and RAH (B) GzmB molecules. The catalytic triad H57, D102, S195 is shown in magenta, and side chains contributing to the substrate-binding pocket (30) are shown in a blue stick representation. The three amino acids that differ between the two proteins (Q/R48, P/A88, and Y/H245, as indicated) are shown in red, and M242 is shown in green. The three mutated amino acids do not affect the structure of the active site or the substrate binding cleft, and are clustered together toward the C-terminal α-helix. (C and D) Contact surface of M242 in QPY (C) and RAH (D) GzmB. Regions of M242 involved in hydrogen bonds are indicated by purple patches. The hydrogen bond formed between M242 and Q48 is not present in RAH GzmB.