Granzyme B Encoded by the Commonly Occurring Human RAH Allele Retains Pro-apoptotic Activity

Abstract

A key function of human granzyme B (GrB) is to induce apoptosis of target cells in conjunction with perforin. The RAH allele is the first documented variant of the human GrB gene, occurs at a frequency of 25-30%, and encodes three amino acid substitutions (Q48R, P88A, and Y245H). It was initially reported that RAH GrB is incapable of inducing apoptosis, but here we show that it has essentially identical proteolytic and cytotoxic properties to wild type GrB. Recombinant RAH and wild type GrB cleave peptide substrates with similar kinetics, are both capable of cleaving Bid and procaspase 3, and are equally inhibited by proteinase inhibitor 9, an endogenous regulator of GrB. Furthermore, cytotoxic lymphocytes from RAH heterozygotes and homozygotes have no defect in target cell killing, and in vitro RAH GrB and wild type GrB kill cells equally well in the presence of perforin. We conclude that the RAH allele represents a neutral polymorphism in the GrB gene.Cytotoxic lymphocytes (CLs) 1 play a central role in cellmediated immunity by destroying virus-infected, foreign, or tumor cells (1). They also contribute to the pathology of autoimmune diseases such as diabetes and to graft versus host disease following bone marrow transplantation (1). On contact with a target cell, the CL releases cytotoxins such as perforin, proteases (granzymes), and death ligands from secretory granules into the intercellular space. Target cell death then follows one of two pathways: activation of death receptors on the target cell surface coupled to caspase activation within the cell or uptake of granzymes in a perforin-dependent manner that leads to caspase activation and intracellular proteolysis (2). In the latter pathway, perforin and granzymes are endocytosed by the target cell, and perforin mediates release of granzymes into the cytoplasm (3). Caspase activation, loss of mitochondrial membrane potential, proteolysis of key house-keeping proteins, DNA degradation, and the disintegration of cellular structures follow rapidly.Human CLs produce granzyme A (GrA) and granzyme B (GrB), two cytotoxic granule serine proteases that activate different death pathways (2). GrA cleaves after basic residues, appears to cause single-stranded DNA breaks, and disrupts repair mechanisms in the target cell, leading to caspase-independent death (4). By contrast, GrB has an unusual preference for cleaving after Asp and is thought to act like an apical caspase in triggering classical apoptosis (2). The role of GrB in cell-mediated immunity is supported by the phenotype of GrBdeficient mice, which produce CLs that cannot induce rapid DNA degradation in target cells, although killing still occurs, possibly via other granzymes (5). Furthermore, apoptosis of transplanted allogeneic hepatocytes in rats is reduced by treatment with GrB inhibitors (6), elevated GrB serum levels occur in several diseases including rheumatoid arthritis, and GrB is implicated in the generation of autoimmune antigens (7).The importance of the granule pathway to CL function is illustra...

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

“…Pfp, Gzm and NK cell cytotoxicity was assessed by 51 Chromium-release assays, sheep red blood cell lysis assays or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as previously described. [37][38][39][40] To assess cell death by timelapse microscopy, target cells were plated in 96-well flat bottom cell culture plates (Grenier Bio-One GmbH, Frickenhausen, Germany) at a density of 0.5 Â 10 4 cells per well and incubated overnight under standard tissue culture conditions. In some experiments, cells were pretreated for 24 h with 100/250 nM taxol, or for 1 h with 2 mM MycB, 10 mM LatB, 5 mM nocodazole or 10 mM blebbistatin.…”

Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species

“…Production of Recombinant Granzymes-Recombinant human granzyme B was expressed and purified from Pichia pastoris and its activity was determined as described previously (25,26). Recombinant, enterokinase activated wild-type GrC and the mutated form of GrC (E192R/E193G) were prepared as described previously (16).…”

“…SLO-mediated Granzyme Cytotoxicity-Cell death mediated by granzymes and recombinant streptolysin-O (SLO) was performed according to (25) and (27).…”

Structure of granzyme C reveals an unusual mechanism of protease autoinhibition

“…GzmC was mutated by PCR mutagenesis with oligonucleotides 5Ј-GGAGCTTCCTTTCGCGAGGATTCTGG and its complement (E192R), 5Ј-GGGAGCTTCCTTCGAAGGGGATTCTGGAGGC and its complement (E193G), and 5Ј-GATCAAGGGAGCTTCCTTCCGCGGGGATTCTGGAGGCCCGC and its complement (E192R/E193G). Electroporation of Pichia pastoris and purification of the gzmC zymogen from culture supernatant were as described (19,20). After activation, enterokinase was removed by cation exchange over SP-Sepharose (GE Healthcare).…”