During granule-mediated killing by cytotoxic T lymphocytes or natural killer cells, the serine protease granzyme B enters the target cell by endocytosis and induces apoptosis. Previous studies suggested a role for the mannose 6-phosphate receptor, but further experiments with purified granzyme B indicated this was not essential. Additionally, it is now clear that grB is exocytosed from killer cells in a high-molecular-weight complex with the proteoglycan serglycin. Here granzyme B was delivered as a purified monomer, or in complex with either glycosaminoglycans or serglycin, and killing was evaluated. When granzyme B was a monomer, soluble mannose 6-phosphate had a limited impact, whereas apoptosis induced by the complexed grB was effectively inhibited by mannose 6-phosphate. Most importantly, when granzyme B and perforin were delivered together from granules, inhibition by mannose 6-phosphate was also observed. In pulldown assays mediated by the cation-independent mannose 6-phosphate receptor, granzyme B bound to the receptor more intensely in the presence of immobilized heparan sulfate. We therefore propose the model that under physiological conditions serglycin-bound granzyme B is critically endocytosed by a mannose 6-phosphate receptor, and receptor binding is enhanced by cell surface heparan sulfate. INTRODUCTIONGranzyme B (grB) is an important effector molecule in the granule-mediated killing pathway by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (Lord et al., 2003;Roberts et al., 2003;. There is intense interest in grB-related mechanisms since killer cell cytotoxicity and thus grB are implicated in both organism homeostasis and pathology, through elimination of virally or oncogenically transformed cells and through autoimmunity or graft versus host disease, respectively (Barry and Bleackley, 2002;Russell and Ley, 2002;Trapani and Smyth, 2002). In the target cell, grB activates an apoptotic pathway by cleaving key substrates in the cytoplasm, though these events are critically codependent on the pore-forming protein perforin (pfn). Because grB must access the substrates within the cell, a key step in grB function must be entry into the target cell.The current hypothesis for grB uptake into the target cell proposes an endocytic mechanism (Froelich et al., 1996), but there has been considerable discussion about this pathway. The first model proposed that the cation-independent mannose 6-phosphate receptor (CI-MPR) was a critical grB receptor (Motyka et al., 2000). However concerns were raised when it was found that grB released from cytotoxic granules was complexed with the large proteoglycan serglycin and that this would be the predominant physiological form of grB (Galvin et al., 1999;Metkar et al., 2002). Notably, uptake studies that identified the CI-MPR as the grB receptor had been performed with a purified free form of grB (Motyka et al., 2000). Because free grB is small (32 kDa; Poe et al., 1991), whereas a grB-serglycin complex is at least eight times larger (serglycin alone from the ...

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the directed exocytosis of lytic effector molecules such as perforin and granzymes. The mechanism by which these proteins enter targets is uncertain. There is ongoing debate over whether the most important endocytic mechanism is nonspecific or is dependent on the cation-independent mannose 6-phosphate receptor. This study tested whether granzyme B endocytosis is facilitated by dynamin, a key factor in many endocytic pathways. Uptake of and killing by the purified granzyme B molecule occurred by both dynamin-dependent and -independent mechanisms. However most importantly, serglycin-bound granzyme B in high-molecular-weight degranulate material from cytotoxic T lymphocytes predominantly followed a dynamin-dependent pathway to kill target cells. Similarly, killing by live cytotoxic T lymphocytes was attenuated by a defect in the dynamin endocytic pathway, and in particular, the pathways characteristically activated by granzyme B were affected. We therefore propose a model where degranulated serglycin-bound granzymes require dynamin for uptake. IntroductionNatural killer cells and cytotoxic T lymphocytes (CTLs) protect a whole organism against dangerous cells, such as those that are infected with virus or are tumorigenic. 1 When natural killers and CTLs recognize a target cell, the latter is killed by either of 2 major pathways: the Fas-Fas ligand (FasL) pathway, or directional exocytosis of membrane-bound cytotoxic granules present in the cytoplasm of the killer cell. The granules mediate the demise of the target cell via the enclosed cytolytic molecules, which include, among others, a family of serine proteases called granzymes, and the pore-forming molecule, perforin (pfn). [2][3][4] The granzymes, via a pfn-dependent mechanism, induce cell death following translocation across the plasma membrane of target cells. Inside the cell, each granzyme fulfills a critical nonoverlapping role to induce apoptosis by cleaving specific subsets of substrates, such as caspases 5,6 and Bid 7-10 by granzyme B (grB), and the SET complex 11 by granzyme A. But in order to achieve cleavage of substrates, clearly a critical first step is the uptake of granzymes into the target cell.The original model of granzyme internalization proposes translocation via a pfn pore in the plasma membrane. However, recent evidence suggests that granzymes are first internalized via endocytosis and then are released into the cytoplasm with the help of pfn by an unknown mechanism. This model is predominantly based on studies performed with grB, which has largely served as the granzyme prototype to date. The first evidence that granzyme uptake occurred by endocytosis was the finding that grB enters cells autonomously. 12-14 Furthermore, grB binds to the cell surface in a concentration-dependent and saturable manner, 12 suggesting a receptor-mediated endocytic mechanism. The grB endocytosis model has further developed with the identification of the cation-independent mannose 6-phosph...

Apoptosis is a potent immune barrier against viral infection, and many viruses, including poxviruses, encode proteins to overcome this defense. Interestingly, the avipoxviruses, which include fowlpox and canarypox virus, are the only poxviruses known to encode proteins with obvious Bcl-2 sequence homology. We previously characterized the fowlpox virus protein FPV039 as a Bcl-2-like antiapoptotic protein that inhibits apoptosis by interacting with and inactivating the proapoptotic cellular protein Bak. However, both Bak and Bax can independently trigger cell death. Thus, to effectively inhibit apoptosis, a number of viruses also inhibit Bax. Here we show that FPV039 inhibited apoptosis induced by Bax overexpression and prevented both the conformational activation of Bax and the subsequent formation of Bax oligomers at the mitochondria, two critical steps in the induction of apoptosis. Additionally, FPV039 interacted with activated Bax in the context of Bax overexpression and virus infection. Importantly, the ability of FPV039 to interact with active Bax and inhibit Bax activity was dependent on the structurally conserved BH3 domain of FPV039, even though this domain possesses little sequence homology to other BH3 domains. FPV039 also inhibited apoptosis induced by the BH3-only proteins, upstream activators of Bak and Bax, despite interacting detectably with only two: BimL and Bik. Collectively, our data suggest that FPV039 inhibits apoptosis by sequestering and inactivating multiple proapoptotic Bcl-2 proteins, including certain BH3-only proteins and both of the critical "gatekeepers" of apoptosis, Bak and Bax.

SERP-1 is a secreted serpin (serine-proteinase inhibitor) encoded by myxoma virus, a poxvirus pathogen of rabbits. SERP-1 is required for myxoma-virus virulence, and the purified protein has been shown to possess independent anti-inflammatory activity in animal models of restenosis and antigen-induced arthritis. As an inhibitor of serine proteinases, SERP-1 acts against tissue-type plasminogen activator, urokinase-type plasminogen activator, plasmin, thrombin and Factor Xa. In the present study, examination of SERP-1 glycosylation-site mutants showed that the N-linked glycosylation of Asn172 was essential for SERP-1 secretion, whereas mutation of Asn99 decreased secretion efficiency, indicating that N-linked glycosylation plays an essential role in the processing and trafficking of SERP-1. Furthermore, comparison of SERP-1 from wild-type myxoma virus and a virus containing a targeted disruption of the MST3N sialyltransferase locus demonstrated that SERP-1 is specifically modified by this myxoma-virus-encoded sialyltransferase, and is thus the first reported viral protein shown to by modified by a virally encoded glycosyltransferase. Sialylation of SERP-1 by the MST3N gene product creates a uniquely charged species of secreted SERP-1 that is distinct from SERP-1 produced from other eukaryotic expression systems, though this has no apparent effect upon the kinetics of in vitro proteinase inhibition. Rather, the role of viral sialylation of SERP-1 likely relates to masking antigenicity or targeting SERP-1 to specific sites of action in vivo.

SERP-1 is a secreted serpin (serine-proteinase inhibitor) encoded by myxoma virus, a poxvirus pathogen of rabbits. SERP-1 is required for myxoma-virus virulence, and the purified protein has been shown to possess independent anti-inflammatory activity in animal models of restenosis and antigen-induced arthritis. As an inhibitor of serine proteinases, SERP-1 acts against tissue-type plasminogen activator, urokinase-type plasminogen activator, plasmin, thrombin and Factor Xa. In the present study, examination of SERP-1 glycosylation-site mutants showed that the N-linked glycosylation of Asn(172) was essential for SERP-1 secretion, whereas mutation of Asn(99) decreased secretion efficiency, indicating that N-linked glycosylation plays an essential role in the processing and trafficking of SERP-1. Furthermore, comparison of SERP-1 from wild-type myxoma virus and a virus containing a targeted disruption of the MST3N sialyltransferase locus demonstrated that SERP-1 is specifically modified by this myxoma-virus-encoded sialyltransferase, and is thus the first reported viral protein shown to by modified by a virally encoded glycosyltransferase. Sialylation of SERP-1 by the MST3N gene product creates a uniquely charged species of secreted SERP-1 that is distinct from SERP-1 produced from other eukaryotic expression systems, though this has no apparent effect upon the kinetics of in vitro proteinase inhibition. Rather, the role of viral sialylation of SERP-1 likely relates to masking antigenicity or targeting SERP-1 to specific sites of action in vivo.