Catherina H. Bird

A Cytosolic Granzyme B Inhibitor Related to the Viral Apoptotic Regulator Cytokine Response Modifier A Is Present in Cytotoxic Lymphocytes

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1′(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 ± 0.3 × 106 M−1 s−1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.

Selective Regulation of Apoptosis: the Cytotoxic Lymphocyte Serpin Proteinase Inhibitor 9 Protects against Granzyme B-Mediated Apoptosis without Perturbing the Fas Cell Death Pathway

ABSTRACT Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.

A Common Fold Mediates Vertebrate Defense and Bacterial Attack

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin

Human granzyme B (GrB) released from cytotoxic lymphocytes plays a key role in the induction of target cell apoptosis when internalized in the presence of perforin. Here we demonstrate that GrB also possesses a potent extracellular matrix remodeling activity. Both native and recombinant GrB caused detachment of immortalized and transformed cell lines, primary endothelial cells, and chondrocytes. Cell detachment by GrB induced endothelial cell death (anoikis). GrB also inhibited tumor cell spreading, migration, and invasion in vitro. Investigation into the underlying mechanism revealed that GrB efficiently cleaves three proteins involved in extracellular matrix structure and function: vitronectin, fibronectin, and laminin. In vitronectin, GrB cleaves after an Arg-Lys-Asp (RGD) motif, which is part of the integrin-binding site found in matrix proteins. We propose that targeting of the integrin-extracellular matrix interface by GrB may allow perforin-independent killing of target cells via anoikis, restrict motility of tumor cells, facilitate lymphocyte migration, or directly reduce virus infectivity. It may also contribute to tissue destruction in diseases in which extracellular GrB is evident, such as rheumatoid arthritis and atherosclerosis.

The major human and mouse granzymes are structurally and functionally divergent.

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even “orthologous” granzymes have species-specific functions, having evolved in distinct environments that pose different challenges.

The Intracellular Granzyme B Inhibitor, Proteinase Inhibitor 9, Is Up-Regulated During Accessory Cell Maturation and Effector Cell Degranulation, and Its Overexpression Enhances CTL Potency

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4+ cells, CD8+ T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8+ T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3−, CD4+, CD8−, CD45+), tonsillar DCs, and DC subsets purified from peripheral blood (CD16+ monocytes and CD123+ plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-α-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.

A New Family of 10 Murine Ovalbumin Serpins Includes Two Homologs of Proteinase Inhibitor 8 and Two Homologs of the Granzyme B Inhibitor (Proteinase Inhibitor 9)

Serine proteinase inhibitors (serpins) are classically regulators of extracellular proteolysis, however, recent evidence suggests that some function intracellularly. Such “ovalbumin” serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B (graB) inhibitor that has an unusual P1 Glu and is present primarily in lymphocytes. In a search for the murine equivalent of PI-9 we screened cDNA libraries, and performed reverse transcriptase-polymerase chain reaction on RNA isolated from leukocyte cell lines and from lymph nodes and spleens of allo-immunized mice. We identified 10 new ovalbumin serpin sequences: two resemble PI-8, two resemble PI-9, and the remaining six have no obvious human counterparts. By RNA analysis only one of the two sequences resembling PI-9 (designated SPI6) is present in mouse lymphocytes while the other (a partial clone designated mBM2A) is predominantly in testis. SPI6 comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to contain the P1-P1′ bond. SPI6 produced in vitro using a coupled transcription/translation system formed an SDS-stable complex with human graB and did not interact with trypsin, chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The second-order rate constant for the interaction was estimated as 8 × 104 m −1 s−1, and inhibition depended on the Glu in the SPI6 reactive center. The SPI6 gene was mapped to the same region on mouse chromosome 13 asSpi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6 is a functional homolog of PI-9, and that the regulation of graB in the mouse may involve a second serpin encoded by mBM2A. Our identification of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin serpins, is consonant with the idea that the larger set of granule and other proteinases known to exist in the mouse (compared with human) is balanced by a larger array of serpins.

Nucleocytoplasmic Distribution of the Ovalbumin Serpin PI-9 Requires a Nonconventional Nuclear Import Pathway and the Export Factor Crm1

ABSTRACT Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (∼70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack

Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu-derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.

Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells.

ABSTRACT Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 reduces accumulation in this intracellular compartment and cytotoxicity two- to threefold. We also show that GrB-mediated cytotoxicity is abrogated by heparin and that target cells deficient in cell surface sulfate or glycosaminoglycans resist GrB. However, heparin does not completely prevent GrB internalization and chondroitin 4-sulfate does not inhibit cytotoxicity, suggesting that glycosaminoglycans are not essential GrB receptors. We propose that GrB enters cells by nonselective adsorptive pinocytosis, exchanging from chondroitin sulfate on serglycin to anionic components of the cell surface. In this electrostatic “exchange-adsorption” model, cs1 and cs2 participate in binding of GrB to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm.