The granzyme B–serglycin complex from cytotoxic granules requires dynamin for endocytosis

Abstract

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...

A Novel Mechanism for Protein Delivery

“…Although the receptor-dependent uptake of the granzyme has been postulated to play a role in granule-mediated apoptosis (3), fluid-phase pinocytosis has been described as a viable mechanism for expanding the number of targets susceptible to granzyme delivery (7,41). Nevertheless, others have shown recently that apoptosis induced by GrB-SG complexes as well as CTLs is entirely dynamin-dependent (18). Therefore, in a physiological setting the relevance of pinocytosis, either constitutive fluid phase pinocytosis or simulated macropinocytosis, as a mechanism for the internalization of GrB remains unclear.…”

“…caveolin-dependent and fluid phase pinocytosis). Previous reports have indicated that granzyme uptake is dynamindependent and -independent (7,18). Internalization pathways may be distinguished by comparing the rate and concentration dependence of uptake.…”

“…Following the original description of specific binding sites for GrB on target cells (3), the granzyme was reported to use the mannose-6-phosphate receptor/insulin-like growth factor-2 receptor (Mpr300) for target cell entry (17). Subsequent work suggested that the uptake occurs both via the dynamin-dependent as well as independent pathways (7,18). Finally, using cell lines derived from Mpr300 and Mpr46 knock-out mice, both receptors were found to be entirely dispensable for CTL-induced apoptosis (19) and allorejection (20).…”

Modulation of the Tumor Cell Phenotype by IFN-γ Results in Resistance of Uveal Melanoma Cells to Granule-Mediated Lysis by Cytotoxic Lymphocytes

“…grB is secreted by CTL in a macromolecular complex bound to serglycin (40). The calcium-independent mannose 6-phosphate receptor (CI-MPR) was shown to act as a receptor for grB (19,41,42), although its importance for granzyme-mediated apoptosis has been questioned (43,44). Other molecules suggested to serve as grB receptors include heat shock protein 70 (45) and CD44 (40,46).…”

Granzyme B-induced apoptosis in cancer cells and its regulation (Review)

“…This GrB receptor-mediated endocytosis model has been further refined with the identification of the cation-independent mannose-6-phosphate receptor (CI-MPR) as a plasma membrane receptor for GrB (124,129,136). The process of the CI-MPR-mediated GrB internalization is clathrin and dynamin-dependent (124,137). Recently, it has been showed that PFN triggers a wounded plasma membrane-repair response, which is the clathrin and dynamin-dependent endocytosis and which removes PFN and granzymes from the plasma membrane to early endosomes (126,132).…”