Granzyme K Displays Highly Restricted Substrate Specificity That Only Partially Overlaps with Granzyme A

Abstract

Granzymes are serine proteases stored in cytolytic granules of cytotoxic lymphocytes that eliminate virus-infected and tumor cells. Little is known about the molecular mechanism and function of granzyme (Gr)K. GrK is similar to GrA in that they are the only granzymes that display tryptase-like activity. Both granzymes induce cell death by single-stranded nicking of the chromosomal DNA by cleaving the same components of the endoplasmic reticulum-associated SET complex. Therefore, GrK may provide a backup and failsafe mechanism for GrA with redundant specificity. In the present study, we addressed the question of whether GrK displays identical substrate specificity as GrA. In peptide-and protease-proteomic screens, GrK and GrA displayed highly restricted substrate specificities that overlapped only partially. Whereas GrK and GrA cleave SET with similar efficiencies likely at the same sites, both granzymes cleaved the pre-mRNA-binding protein heterogeneous ribonuclear protein K with different kinetics at distinct sites. GrK was markedly more efficient in cleaving heterogeneous ribonuclear protein K than GrA. GrK, but not GrA, cleaved the microtubule network protein ␤-tubulin after two distinct Arg residues. Neither GrK cleavage sites in ␤-tubulin nor a peptidebased proteomic screen revealed a clear GrK consensus sequence around the P1 residue, suggesting that GrK specificity depends on electrostatic interactions between exosites of the substrate and the enzyme. We hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death but that it also displays a unique function by cleaving its own specific substrates.Important players in the immune defense against tumor cells and virus-infected cells are cytotoxic T lymphocytes and natural killer cells (1, 2). These immune cells predominantly destroy their target cells by releasing the content of their cytolytic granules, containing the pore-forming protein perforin and a set of serine proteases known as granzymes. In humans, five different granzymes (GrA, GrB, GrH, GrK, and GrM) 2 have been identified that all induce cell death by cleaving critical intracellular substrates. Although GrA and GrB have been extensively studied, little is known about the functions and mechanisms of the other granzymes.The GrA cell death pathway is characterized by singlestranded DNA damage, apoptotic morphology, mitochondrial dysfunction, and loss of cell membrane integrity and occurs independent of caspases and the GrB-induced apoptotic routes (1-5). GrA is targeted inside the mitochondrion (6), where it triggers an increase in reactive oxygen species and loss of transmembrane potential (3, 5). After mitochondrial damage, GrA targets a 270 -440-kDa endoplasmic reticulum-associated complex (SET complex) that contains three GrA substrates, i.e. nucleosome assembly protein SET (4), DNA-binding protein HMG-2 (7), and base excision repair enzyme Ape1 (8). Cleavage of SET by GrA allows the SET complex component DNase NM23H1 to make single-stran...

Mouse granzyme K has pro-inflammatory potential

“…As expected, only Tc cells from LCMV-immune B6 but not from gzmAxB À/À and perfxgzmAxB À/À mice expressed gzmA and gzmB proteins (data not shown). (Mo)gzmK manifested enzymatic activity and to verify their predicted differences in cleavage specificity, 12 their amidolytic activities were compared against four different chromogenic substrates. Both gzms were proteolytically active, with each exhibiting distinct preferences for the four related chromogenic substrates, especially Tos-Gly-pro-ArgpNA (Table 1).…”

“…12 Similar to (Hu)gzmA, the original report indicated that isolated Rat and (Hu)gzmKs are cytotoxic in vitro. 13 A more recent study has suggested that isolated (Hu)gzmK induces biochemical events similar to (Hu)gzmA in target cells.…”

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication

“…21,34 Interestingly, many members of the hnRNP protein family have also been identified as potential substrates of other granzymes than GrM. [35][36][37][38] Further studies It remains an intriguing question why GrM targets viral pp71 17 and host cell hnRNP K to attack the initial IE events of HCMV replication rather than just killing HCMV-infected cells. Consistent with our findings, IE1-and pp65-specific CD4 þ T cells and CD8 þ T cells can recognize and kill IE1 and pp65 peptide-loaded target cells, but fail to induce cell death in HCMV-infected target cells after recognition and degranulation, whereas HCMV replication is efficiently inhibited 39,40 (Dr. MR Wills, personal communication (Department of Medicine, University of Cambridge, Cambridge, UK)).…”

Testosterone regulates granzyme K expression in rat testes

“…Considering the above mentioning and results of this study, GZMK may play a crucial role in facilitating the degradation of tubulobular structures and translocation of spermatids. Th e tubulobular structures in testes are made by microtubules , and GZMK, a tryptase, specifi cally targets of β-tubulin (Bovenschen et al 2009). Moreover, microtubules are abundant in the Sertoli cell cytoplasm, are oriented in the direction of the spermatid translocation and surround the cisterns where spermatids remain embedded (Redenbach et al 1992;Vogl et al 2000).…”

“…Alternately, GZMK may also initiate germ cell apoptosis and have a role in the degradation of the apoptotic bodies in testes. GZMK induce apoptosis independently of the caspase-mediated pathways through the nuclear morphological changes and single-strand DNA breaks (Bovenschen et al 2009). Even though, GZMK staining was absent in the nuclei of the same germ cell stages in the seminiferous tubule that showed apoptosis aft er EDS treatment.…”