Granzyme B

Granzyme B (GzmB, EC 3.4.21.79) as one of the most abundant members of the granzyme family in humans and mice is a caspase-like serine protease that is encoded by the GZMB gene, expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells along with the pore forming protein perforin to mediate apoptosis in target cells. GzmB has also more recently found to be produced by a wide range of non-cytotoxic cells ranging from basophils and mast cells to smooth muscle cells. GzmB is crucial for the rapid induction of target cell apoptosis by CTL in cell-mediated immune response and could cut substrates post-aspartic acid residues. GzmB has been evidenced to cause double-stranded DNA fragmentation and caspase-dependent or -independent cell death.

GzmB also shows synergistic action in viral clearance, while it is not sufficient for killing a wide variety of viruses against infections. In addition, GzmB involves in extracellular matrix remodeling and inflammation by stimulating cytokine release. The elevation of GzmB levels is implicated in some autoimmune diseases, several skin diseases, and type 1 diabetes.

Molecular Structure

In humans, GzmB encoded by GZMB on chromosome 14q.11.2 is 3.2kb and consists of 5 exons, which is thought to evolve from a granzyme H related precursor and is more effective at lower concentrations in comparison with the other granzymes. In GzmB structure, two 6 stranded β sheets and 3 trans-domain segments can be found. In the granules of cytotoxic lymphocytes the enzyme can exist in two glycosylated forms. GzmB containing a catalytic triad histidine-aspartic acid-serine in its active site would preferentially cleave after the location of an aspartic acid residue in the P1 position. The aspartic acid residue to be cleaved connects with an arginine residue in the enzyme's binding pocket. GzmB is generally active at a neutral pH and therefore it behaves inactive in the acidic CTL granules. The enzyme is also deactivated when bound with serglycin in the granules to avoid apoptosis of cytotoxic T cells. Initially, GzmB is in a form of inactive precursor zymogen with an additional amino terminal peptide sequence, which can be cleaved by cathepsin C by removing two amino acids. GzmB could be also activated by cathepsin H.

Delivery

GzmB is released with perforin with a radius of 5.5 nm that can insert into the plasma membrane of a target cell by forming a pore. GzmB has a smaller stokes radius of 2.5 nm than perforin and thus can pass through the pore of perforin into the target to be destroyed. Alternatively, once released, GzmB binds with negatively charged heparan sulphate containing receptors on a target cell and is endocytosed. Subsequently, the vesicles carrying the enzyme inside burst and expose GzmB to the cytoplasm and its substrates. Hsp-70 also assists the entry of GzmB. It is also proposed that GzmB enters a target by first exchanging its bound serglycin for negative phospholipids in a target's plasma membrane and then entry appears in the less selective process of absorptive pinocytosis.

Granzyme B Mediated Apoptosis

GzmB can cleave caspases 8 and 10 once inside the target cell and activate initiator executioner caspases 3 and 7 that trigger apoptosis. Caspase 7 is the most susceptible to GzmB, and caspases 3, 8, and 10 are only cleaved to intermediate fragments and need additional cleavage for full activation. GzmB can destroy BID that causes BAX/BAK oligomerisation and releases cytochrome c from the mitochondria and also split ICAD that results in DNA fragmentation and the laddering pattern associated with apoptosis. GzmB has a potential of over 300 substrates and can break Mcl-1 down in the outer mitochondrial membrane to relieve its inhibition of Bim, which stimulates BAX/BAK oligomerisation, mitochondrial membrane permeability and apoptosis. Hs-1 associated protein X-1 can be also cleaved by GzmB to facilitate mitochondria polarisation. GzmB can also mediate cell death through generating a cytotoxic level of mitochondrial reactive oxygen species. The caspase independent pathways of cell death are thought to be capable of defeating viruses that inhibit caspases and prevent apoptosis.

Targets

Many substrates of GzmB are located in the nucleus. Poly ADP ribose polymerase and DNA protein kinase could be cleaved by GzmB to block DNA repair and retroviral DNA integration. Nucleophosmin, topoisomerase 1 and nucleolin can also be destructed by GzmB so as to prevent viral replication. ICP4 as an essential protein from the HSV 1 virus used for gene transactivation and muclear mitotic apparatus protein can be cracked to prevent mitosis. GzmB can also cut DNA binding protein into a 50 kDa fragment and then into an additional 60 kDa moiety indirectly through the caspases it activates. Furthermore, Granzyme B can degrade many proteins in the extracellular matrix such as fibronectin, vitronectin and aggrecan, and this cleavage can further cause cell death by anoikis and release alarmins from the extracellular matrix inducing inflammation. GzmB secreted by Basophils also degrades endothelial cell-cell contacts that allow extravasation to sites of inflammation. The cleavage of vitronectin occuring at the RGD integrin binding site could interrupt cell growth signalling pathways and the degradation of laminin, and fibronectin interrupts dermal-epidermal junction attachment and cross talk, while decorin destruction by GzmB leads to collagen disorganisation, skin thinning and aging. Keratinocytes can express GzmB after exposure to UVA and UVB that associates with photoaging of the skin. The breakage of the von Willebrand factor and plasminogen inhibits platelet aggregation and produces an angiostatin fragment preventing angiogenesis, respectively. The cutting of fibronectins and vitronectins delays the formation of a provisional matrix and thus impairs wound healing.

Roles in Diseases

GzmB has a normal concentration ranging from 20 to about 40 pg/mL in blood plasma and retains almost 70% activity. The increase in concentrations of GzmB is related to a number of disease states. GzmB generates autoantigens by cleaving in disordered regions and linker regions of antigens that exposes new epitopes, which then causes the development of autoimmune diseases. The release of GzmB with perforin from CD8 T cells also causes heart and kidney transplant rejection through killing of allogeneic endothelial cells. The destruction of insulin that produces β cells in pancreatic islets mediated by T cells and GzmB contributes to type 1 diabetes. GzmB also mediates the death of cells after spinal cord injury and is found with elevated levels in rheumatoid arthritis. GzmB secreted from NK and T cells makes contributions to the apoptosis of bronchial epithelial cells and thus causes chronic obstructive pulmonary disease. The destabilisation and remodeling of matrix by GzmB are connected with asthma pathogenesis. Vitiligo, contact dermatitis, lichen sclerosus, and lichen planus cases can be resulted from GzmB by killing melanocytes and its overexpression. Cytotoxic cells expressing GzmB have close relationship with hair follicles which plays a possible role in hair loss. The ECM remodelling properties of GzmB have also implicated in left ventricular remodelling, which increases the subsequent possibilities of myocardial infarction.