Granzyme

Granzymes are serine proteases that are released by cytoplasmic granules within cytotoxic T cells and natural killer (NK) cells. They induce programmed cell death in the target cell, thus eliminating cells that have become cancerous or are infected with viruses or bacteria. The granzymes also kill bacteria and inhibit viral replication. In NK cells and T cells, the granzymes are packaged in cytotoxic granules to prevent harm to the host cell. Other locations that granzymes can be detected are in the rough endoplasmic reticulum, golgi complex, and the trans-golgi reticulum. The contents of the cytotoxic granules function to permit entry of the granzymes into the target cell cytosol. Granzymes are identified as being part of the serine esterase family. They are closely related to other immune serine proteases expressed by innate immune cells, such as neutrophil elastase and cathepsin G.

Cytotoxic T cells and natural killer cells release a protein called perforin, which attacks the target cells. Researchers used to think that perforin creates pores within the cell membranes, through which the granzymes can enter, inducing apoptosis. However, new evidence indicates that a multimeric complex (granzyme B, perforin, and granulysin) can enter a cell through the mannose 6-phosphate receptor (or another receptor found in tumor cells) and is enclosed in a vesicle (a sac). Not mentioned in reference Perforin then allows GrB to pass through the vesicle surface and into the cell, causing apoptosis by various pathways.

They do so by cleaving caspases (especially caspase-3), which in turn activates caspase-activated DNase. This enzyme degrades DNA, thus inducing apoptotic cascades. Also, GrB cleaves the protein Bid, which recruits the protein Bax and Bak to change the membrane permeability of the mitochondria, causing the release of cytochrome c (which is one of the parts needed to activate caspase-9 via the apoptosome), Smac/Diablo and Omi/HtrA2 (which suppress the inhibitor of apoptosis proteins (IAPs)), among other proteins. As well, GrB is shown to cleave many of the proteins responsible for apoptosis without the aid of caspase, as proven by experiments on caspase knockout mice CTL cells incubated with other cells.

In addition to killing their target cells, granzymes can target and kill intracellular pathogens. Granzymes A and B induce lethal oxidative damage in bacteria by cleaving components of the electron transport chain, while granzyme B cleaves viral proteins to inhibit viral activation and replication. The granzymes bind directly to the nucleic acids DNA and RNA, this enhances their cleavage of nucleic acid binding proteins. Nucleic acid binding may explain the localization of the granzymes to the nuclei of their target cells.

Other Granzyme Functions

In Cullen's paper “Granzymes in Cancer and Immunity” he discusses how granzyme A has been known to be found in elevated levels within patients who currently have an infectious disease and/or in a pro-inflammatory state. Granzymes have also been found to help initiate the inflammatory response. “For example, rheumatoid arthritis patients have increased levels of granzyme A in the synovial fluid of swollen joints”. When granzymes are in an extracellular state they have the ability to activate macrophages and mast cells to initiate the inflammatory response. The interaction between the granzymes and somatic cells are still unexplainable but advances in understanding the process are being made constantly. Other granzymes like granzyme K have been found in high levels of patients who have gone septic. Granzyme H has been found to have a direct correlation with patients who have a viral infection. Scientists are able to conclude that granzyme H specializes in detecting ‘proteolytic degradation’ which is found in viral proteins.

Cullen further states in his paper that granzymes may have a role in immunomodulation, or the job of maintaining homeostasis in the immune system during an infection. “In humans, loss of perforin function leads to a syndrome called familial hemophagocytic lymphohistiocytosis […]”. This syndrome can lead to death because both T cells and macrophages grown to fight the pathogen. This growth leads to inflammation of vital organs and can potentially lead to death.

In Trapani’s paper he talks about how granzymes may have other functions, in addition to their ability to fight off infection. Granzyme A contains certain chemicals that allow it to cause proliferation in B cells to reduce the chance of cancer growth and formation. Test on mice have shown that granzyme A and B might not have a direct link to controlling viral infections, but helping accelerate the immune systems response.

History

In 1986 Jürg Tschopp and his group published a paper on their discovery of granzymes. In the paper they discussed how they purified, characterized and discovered a variety of granzymes found within cytolytic granules that were carried by cytotoxic T lymphocytes and natural killer cells. Jürg was able to identify 8 different granzymes and discovered partial amino acid sequences for each. The molecules were unofficially named Grs for five years before Jürg and his team came up with the name granzymes which was widely accepted by the scientific community.

Granzyme secretion can be detected and measured using Western Blot or ELISA techniques. Granzyme secreting cells can be identified and quantified by flow cytometry or ELISPOT. Alternatively, granzyme activity can be assayed by virtue of their protease activity.

Granzymes in Cancer research

In Cullen's paper “Granzymes in Cancer and Immunity” he describes the process of “immune surveillance [as] the process whereby precancerous and malignant cells are recognized by the immune system as damaged and are consequently targeted for elimination”. For a tumor to progress it requires conditions within the body and surrounding area to be growth-promoting. Almost all people have suitable immune cells to fight off tumors in the body. Studies have shown that the immune system even has the ability to prevent precancerous cells from growing and arbitrate the regression of established tumors. The dangerous thing about cancer cells is they have the ability to inhibit the function of the immune system. Although a tumor may be in its beginning stage and very weak, it may be giving off chemicals that inhibit the function of the immune system allowing it to grow and become harmful. Tests have shown that mice without granzymes and perforins are at high risk to have tumors spread throughout their body.

Tumors have the ability to escape from immune surveillance by secreting immunosuppressive TGF-β. This inhibits proliferation and activation of T cells. TGF-β production is the most potent mechanism of immune avoidance used by tumors. TGF-β inhibits expression of five different cytotoxic genes including perforin, granzyme A, and granzyme B, which then inhibits T cell-mediated tumor clearance.

Perforin’s role in protecting the body against lymphoma was emphasized when scientists discovered that p53 did not have as big of a role in lymphoma surveillance as its counterpart perforin. Perforin and granzymes have been found to have a directly related ability to protect the body against the formation of different kinds of lymphomas.

Genes

• GZMA
• GZMB
• GZMH
• GZMK
• GZMM