Granzyme-mediated cytotoxicity does not involve the mannose 6-phosphate receptors on target cells

Cytotoxic T lymphocytes (CTL) and natural killer cells secrete granzymes to kill infected or transformed cells. The mannose 6-phosphate receptor (Mpr) 300 on target cells has been reported to function as receptor for secreted granzyme B. Using lymphoblasts and mouse embryonal fibroblast lines from Mpr300 and Mpr46 knockout mice, we show here that both receptors are not essential for CTL-induced apoptosis. Similarly, cells exposed to either monomeric granzyme B or granzyme B-serglycin complexes readily internalize the granzyme and undergo apoptosis in the absence of Mpr300 and Mpr46. Further, no colocalization of granzyme B and Mpr300 could be observed in target cells after internalization. In conclusion, these results strongly argue against an Mpr300- or Mpr46-dependent pathway of granzyme-mediated killing and provide new insight in the internalization of monomeric and complexed granzyme B.     

Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis

The serine proteinase granzyme B is crucial for the rapid induction of target cell apoptosis by cytotoxic T cells. Granzyme B was recently demonstrated to enter cells in a perforin-independent manner, thus predicting the existence of a cell surface receptor(s). We now present evidence that this receptor is the cation-independent mannose 6-phosphate/insulin-like growth factor receptor (CI-MPR). Inhibition of the granzyme B-CI-MPR interaction prevented granzyme B cell surface binding, uptake, and the induction of apoptosis. Significantly, expression of the CI-MPR was essential for cytotoxic T cell-mediated apoptosis of target cells in vitro and for the rejection of allogeneic cells in vivo. These results suggest a novel target for immunotherapy and a potential mechanism used by tumors for immune evasion.     

Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo

Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro.     

Low and high affinity receptors mediate cellular uptake of heparanase

Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteometrade mark GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors co-operate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated.     

Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.     

Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction

Killer lymphocytes utilize the synergy of a membranolytic protein, perforin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined. We have previously shown that perforin specifically induces the redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has not been demonstrated. In the present study, we used confocal laser scanning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptosis. Two distinct and mutually exclusive cellular responses were observed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FITC-grB in the nucleus (maximal at 7 min; t1/2 of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cytoplasm, and escaped apoptosis. Under these conditions, DNA fragmentation was not observed for at least 13 min, indicating nuclear accumulation of grB preceded the execution phase of apoptosis. Furthermore, nuclear import of grB proceeded through an intact nuclear membrane, as the nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dextran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB. These findings indicated that perforin-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation. The cell membrane lesions formed by perforin in these experiments were not large enough to permit a 13 kDa protein (yeast cdk p13suc) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equilibrate between the cytosol and the exterior. Therefore, transmembrane pores large enough to allow passive diffusion of grB (32 kDa) into the cell are not necessary for apoptosis. Rather, a perforin-dependent signal results in a redistribution of grB from the cytoplasm to the nucleus, where it may contribute to the nuclear changes associated with apoptosis.     

A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and internalization assays, granzyme B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced by purified granzyme B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of granzyme B from serglycin to cell surface proteins.     

Cell surface-expressed cation-independent mannose 6-phosphate receptor (CD222) binds enzymatically active heparanase independently of mannose 6-phosphate to promote extracellular matrix degradation

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate, an important structural component of the extracellular matrix (ECM) and vascular basement membrane (BM). The cleavage of heparan sulfate by heparanase-expressing cells, such as activated leukocytes, metastatic tumor cells, and proliferating endothelial cells, facilitates degradation of the ECM/BM to promote cell invasion associated with inflammation, tumor metastasis, and angiogenesis. In addition to its enzymatic function, heparanase has also recently been shown to act as a cell adhesion and/or signaling molecule upon interaction with cell surfaces. Despite the obvious importance of the mechanisms for the binding of heparanase to cell surfaces, the receptor(s) for heparanase remain poorly defined. In this study, we identify the 300-kDa cation-independent mannose 6-phosphate receptor (CIMPR) as a cell surface receptor for heparanase. Purified platelet heparanase was shown to bind the human CIMPR expressed on the surface of a transfected mouse L cell line. Optimal binding was determined to be at a slightly acidic pH (6.5-7.0) with heparanase remaining on the cell surface for up to 10 min at 37 degrees C. In contrast, mouse L cells or Chinese hamster ovary cells expressing the cation-dependent mannose 6-phosphate receptor (CDMPR) showed no binding of heparanase. Interestingly, the binding of heparanase to CIMPR was independent of Man-6-P moieties. Significantly, primary human T cells upon activation were shown to dramatically up-regulate levels of cell surface-expressed CIMPR, which showed a concomitant increase in their capacity to bind heparanase. Furthermore, the tethering of heparanase to the surface of cells via CIMPR was found to increase their capacity to degrade an ECM or a reconstituted BM. These data suggest an important role for CIMPR in the cell surface presentation of enzymatically active heparanase for the efficient passage of T cells into an inflammatory site and have implications for the use of this mechanism by other cell types to enhance cell invasion.     

Cytosolic delivery of granzyme B by bacterial toxins: evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.     

Natural killer cell-mediated cytotoxicity does not depend on recognition of mannose 6-phosphate residues

Interaction of mannose 6-phosphate-specific receptors with their ligands has been suggested to be essential for natural killer cell (NK)-mediated cytotoxicity. Indeed, mannose 6-phosphate-specific receptors and ligands bearing mannose 6-phosphate residues are demonstrable on human peripheral blood leukocytes with NK activity as well as on K-562 NK target cells, allowing at least in principle such an interaction. It can also be shown that NK activity of human peripheral blood leukocytes is inhibited by mannose 6-phosphate. The following observations, however, exclude an essential role of the mannose 6-phosphate receptor-ligand system in NK cell-mediated cytotoxicity. 1) NK cytotoxicity is sensitive to a broad range of structurally unrelated sugar phosphates. 2) NK activity is normal in patients with I cell disease (mucolipidosis II), which due to a genetic defect are unable to synthesize the ligands for the mannose 6-phosphate-specific receptor. 3) NK cytotoxicity is not inhibited by an antiserum against the mannose 6-phosphate receptor, which blocks the receptor function.     

HSV and glycoprotein J inhibit caspase activation and apoptosis induced by granzyme B or Fas

HSV-1 inhibits apoptosis of infected cells, presumably to ensure that the infected cell survives long enough to allow completion of viral replication. Because cytotoxic lymphocytes kill their targets via the induction of apoptosis, protection from apoptosis could constitute a mechanism of immune evasion for HSV. Several HSV genes are involved in the inhibition of apoptosis, including Us5, which encodes glycoprotein J (gJ). Viruses deleted for Us5 showed defects in inhibition of caspase activation after Fas ligation or UV irradiation. Transfected cells expressing the Us5 gene product gJ were protected from Fas- or UV-induced apoptosis, as measured by morphology, caspase activation, membrane permeability changes, or mitochondrial transmembrane potential. In contrast, caspase 3 activation in mitochondria-free cell lysates by granzyme (gr)B was inhibited equivalently by Us5 deletion and rescue viruses, suggesting that gJ is not required for HSV to inhibition this process. However, mitochondria-free lysates from transfected cells expressing Us5/gJ were protected from grB-induced caspase activation, suggesting that Us5/gJ is sufficient to inhibit this process. Transfected cells expressing Us5/gJ were also protected from death induced by incubation with purified grB and perforin. These findings suggest that HSV has a comprehensive set of immune evasion functions that antagonize both Fas ligand- and grB-mediated pathways of CTL-induced apoptosis. The understanding of HSV effects on killing by CTL effector mechanisms may shed light on the incomplete control of HSV infections by the immune system and may allow more rational approaches to the development of immune modulatory treatments for HSV infection.     

Interchain proteolysis, in the absence of a dimerization stimulus, can initiate apoptosis-associated caspase-8 activation

Caspases coordinate the internal demolition of the cell that is seen during apoptosis. Proteolytic processing of caspases is observed during apoptosis, and this correlates with conversion of inactive caspase proenzymes into their active two-chain forms. However, recent studies have suggested that caspase-8 is activated through dimerization and that interchain proteolysis is not sufficient for activation of this caspase. This proposal casts doubt upon whether caspase-8 is productively activated by granzyme B during granule-dependent cytotoxic T lymphocyte or natural killer cell-mediated killing, for example. Contrary to the dimerization model, we show that direct proteolysis of caspase-8 by the cytotoxic T lymphocyte protease granzyme B, or by caspase-6, produces an active enzyme that displays robust proteolytic activity toward synthetic as well as natural caspase-8 substrates. These data suggest that enforced dimerization of caspase-8 zymogens by scaffold proteins such as Fas-associated protein with death domain (FADD), although important in certain contexts, is not a prerequisite for activation of this protease.     

The proteoglycan repertoire of lymphoid cells

Proteoglycans have been studied to a limited extent in lymphoid cells. In this study we have investigated the expression of proteoglycans in B-cells, CD4+ T-cells, CD8+ T-cells, natural killer cells, as well as in nine different cell lines established from patients with lymphoid malignancies. Serglycin was the major proteoglycan expressed at mRNA level by the primary lymphocytes. None of the syndecans or glycpicans was detected at mRNA level in the primary lymphocytes, except for syndecan-4 in CD4+ T-cells and CD8+ T-cells. All lymphoid cell lines expressed serglycin mRNA, as well as one or several members of the syndecan and glypican families. Further, increased synthesis of proteoglycans was found in the cell lines compared to the primary lymphocytes, as well as the presence of heparan sulfate on the cell surface of five of the cells lines. Western blot analysis showed a close correlation between serglycin mRNA level and expression of serglycin core protein. Our results show that serglycin is a major proteoglycan in all the normal lymphoid cells and that these cells carry little, or none, proteoglycans on the cell surface. Serglycin was also a major proteoglycan in the malignant lymphoid cells, but these also expressed one or more types of cell surface proteoglycans. Thus, malignant transformation of lymphoid cells may be followed by increased synthesis of proteoglycans and expression of cell surface proteoglycans.     

A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.     

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 μg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.     

Imaging of lytic granule exocytosis in CD8+ cytotoxic T lymphocytes reveals a modified form of full fusion

Here we imaged the exocytosis of lytic granules from human CD8⁺ cytotoxic T lymphocytes using rapid total internal reflection microscopy, Lamp-1 tagged with mGFP to follow the fate of the lytic granule membrane, and granzyme A, granzyme B or serglycin tagged with mRFP to follow the fate of lytic granule cargo. Lytic granules were released by full fusion with the plasma membrane, such that the entire granule content for all three cargos visualized was released on a subsecond time scale. The behavior of GFP-Lamp-1 was, however, more complex. While it entered the plasma membrane in all cases, the extent to which it then diffused away from the site of exocytosis varied from nearly complete to highly restricted. Finally, the diffusion properties upon release of the three cargos examined put an upper limit on the size of the macromolecular complex of granzyme and serglycin that is presented to the target cell.     

Heparan sulfate is essential for high mobility group protein 1 (HMGB1) signaling by the receptor for advanced glycation end products (RAGE)

In a proteomic search for heparan sulfate-binding proteins on monocytes, we identified HMGB1 (high mobility group protein B1). The extracellular role of HMGB1 as a cytokine has been studied intensively and shown to be important as a danger-associated molecular pattern protein. Here, we report that the activity of HMGB1 depends on heparan sulfate. Binding and competition studies demonstrate that HMGB1 interacts with CHO and endothelial cell heparan sulfate. By site-directed mutagenesis, we identified a loop region that connects the A-box and B-box domains of HMGB1 as responsible for heparan sulfate binding. HMGB1-induced Erk1/2 and p38 phosphorylation is abolished when endothelial heparan sulfate is removed or blocked pharmacologically, resulting in decreased HMGB1-induced endothelial sprouting. However, mutated HMGB1 that lacks the heparan sulfate-binding site retained its signaling activity. We show the major receptor for HMGB1, receptor for advanced glycation end products (RAGE), also binds to heparan sulfate and that RAGE and heparan sulfate forms a complex. Our data establishes that the functional receptor for HMGB1 consists of a complex of RAGE and cell surface heparan sulfate.     

Cellular uptake of mammalian heparanase precursor involves low density lipoprotein receptor-related proteins, mannose 6-phosphate receptors, and heparan sulfate proteoglycans

Mammalian heparanase, strongly implicated in the regulation of cell growth, migration, and differentiation, plays a crucial role in inflammation, angiogenesis, and metastasis. There is thus a clear need for understanding how heparanase activity is regulated. Cells can generate an active form of the enzyme from a larger inactive precursor protein by a process of secretion-recapture, internalization, and proteolytic processing in late endosomes/lysosomes. Cell surface heparan sulfate proteoglycans are the sole known components with a role in this trafficking of the heparanase precursor. Here, we provide evidence that heparan sulfate proteoglycans are not strictly required for this process. More importantly, by heparanase transfection, binding, and uptake experiments and by using a combination of specific inhibitors and receptor-defective cells, we have identified low density lipoprotein receptor-related proteins and mannose 6-phosphate receptors as key elements of the receptor system that mediates the capture of secreted heparanase precursor and its trafficking to the intracellular site of processing/activation.     

Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in target cells

Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed granulysin transgenic (GNLY(+/-)) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant granulysin and cell-delivered granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.     

Ligand interactions of the cation-dependent mannose 6-phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor

The interactions of the bovine cation-dependent mannose 6-phosphate receptor with monovalent and divalent ligands have been studied by equilibrium dialysis. This receptor appears to be a homodimer or a tetramer. Each mole of receptor monomer bound 1.2 mol of the monovalent ligands, mannose 6-phosphate and pentamannose phosphate with Kd values of 8 X 10(-6) M and 6 X 10(-6) M, respectively and 0.5 mol of the divalent ligand, a high mannose oligosaccharide with two phosphomonoesters, with a Kd of 2 X 10(-7) M. When Mn2+ was replaced by EDTA in the dialysis buffer, the Kd for pentamannose phosphate was 2.5 X 10(-5) M. By measuring the affinity of the cation-dependent and cation-independent mannose 6-phosphate receptors for a variety of mannose 6-phosphate analogs, we conclude that the 6-phosphate and the 2-hydroxyl of mannose 6-phosphate each contribute approximately 4-5 kcal/mol of Gibb's free energy to the binding reaction. Neither receptor appears to interact substantially with the anomeric oxygen of mannose 6-phosphate. The receptors differ in that the cation-dependent receptor displays no detectable affinity for N-acetylglucosamine 1'-(alpha-D-methylmannopyranose 6-monophosphate) whereas this ligand binds to the cation-independent receptor with a poor, but readily measurable Kd of about 0.1 mM. The spacing of the mannose 6-phosphate-binding sites relative to each other may also differ for the two receptors.