Membrane receptors are not required to deliver granzyme B during killer cell attack

Abstract

Granzyme B (GzmB), a serine protease of cytotoxic T lymphocytes and natural killer (NK) cells, induces apoptosis by caspase activation after crossing the plasma membrane of target cells. The mechanism of this translocation during killer cell attack, however, is not understood. Killer cells release GzmB and the membrane-disturbing perforin at the contact site after target recognition. Receptor-mediated import of glycosylated GzmB and release from endosomes were suggested, but the role of the cation-independent mannose 6-phosphate receptor was recently refuted. Using recombinant nonglycosylated GzmB, we observed binding of GzmB to cellular membranes in a cell type-dependent manner. The basis and functional impact of surface binding were clarified. GzmB binding was correlated with the surface density of heparan sulfate chains, was eliminated on treatment of target cells with heparinase III or sodium chlorate, and was completely blocked by an excess of catalytically inactive GzmB or GzmK. Although heparan sulfate-bound GzmB was taken up rapidly into intracellular lysosomal compartments, neither of the treatments had an inhibitory influence on apoptosis induced by externally added streptolysin O and GzmB or by natural killer cells. We conclude that membrane receptors for GzmB on target cells are not crucial for killer cell-mediated apoptosis. IntroductionGranzymes are a family of granular serine proteases expressed by cytotoxic T lymphocytes (CTLs) and NK cells implicated in immune defense reactions. Granzyme B (GzmB), the most prominent member of this family, induces apoptosis of target cells after cytosolic delivery by caspase-dependent and -independent pathways, resulting in the activation of effector caspases and mitochondrial depolarization, respectively. 1,2 The mechanism of translocation across the plasma membrane, however, is poorly understood. In vivo, the apoptotic functions of granzymes strictly depend on the membrane-binding protein perforin. Mice without the functional perforin gene display a strong reduction of granular cytotoxicity. [3][4][5][6][7] Although perforin can form lytic pores across membranes at high concentrations, such as the structurally related C9 component of the terminal complement complex, 8 sublytic concentrations of perforin already synergize with granzymes in the absence of transmembrane pores. 9,10 In vitro, the sublytic activity of perforin can be replaced by other pore-forming proteins, such as streptolysin O (SLO) and pneumolysin. 9 The latter agents also cooperate in an unknown manner with GzmB at sublytic concentrations without generating pores for the delivery of proteins in the size range of granzymes. 9 Although membrane-bound perforin is able to generate nonspecific calcium channels but no open pores across the membranes of nucleated target cells, it is difficult to understand how macromolecules of 25 kDa, such as nonglycosylated GzmB, can reach the cytosol. Endocytosis and redistribution of externally added GzmB inside the target cell occurs rapidly, within 15 minutes, ...

Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death

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A Hydrophobic Patch on Proteinase 3, the Target of Autoantibodies in Wegener Granulomatosis, Mediates Membrane Binding via NB1 Receptors

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A Novel Mechanism for Protein Delivery

“…Recent work has suggested that the level of HSPGs on target cells may not be associated with susceptibility to GrBinduced apoptosis (40). Nevertheless, the authors of the same study concluded that HSPGs are required for GrB binding and uptake by target cells.…”

“…Because xyloside treatment reduces the level of both HS and CS GAG expression, the results suggest that both GAGs participate in a GrB uptake that contributes to cell-mediated cytotoxicity. Kurschus et al relied on enzymatic removal of HS GAGs with heparinase, monitoring depletion with an anti-N-sulfated glucosamine antibody (40). This limited treatment may not have effectively removed GrB-binding GAGs, either HS and/or CS, below a threshold that prevented induction of cell death.…”