Granzyme B delivery via perforin is restricted by size, but not by heparan sulfate-dependent endocytosis

Abstract

How granzymes gain entry into the cytosol of target cells during killer cell attack has been the subject of several studies in the past, but the effective delivery mechanism during target cell encounter has not been clarified. Here we show that granzyme B (GzmB) mutants lacking binding to negatively charged, essentially heparan-sulfate-containing membrane receptors are poorly endocytosed yet are delivered to the cytosol with efficacy similar to that of WT GzmB. In a cell-based system GzmB-deficient natural killer cells provided perforin (pfn) by natural polarized secretion and synergized with externally added GzmB. Whereas receptor (heparan sulfate)-dependent endocytosis was dispensable, delivery of larger cargo like that of GzmB fusion proteins and GzmB-antibody complexes was restricted by their size. Our data support the model in which granzymes are primarily translocated through repairable membrane pores of finite size and not by the disruption of endocytosed vesicles. We conclude that structurally related translocators, i.e., perforin and cholesterol-dependent cytolysins, deliver deathly cargo across host cell membranes in a similar manner.

Fig. 1.

Strongly diminished heparan sulfate binding and endocytosis of GzmB mutants. (A) Schematic representation of WT-GzmB and the three variants that were expressed and produced in active form (SI Materials and Methods). Sequences around the mutated positions are shown in single-letter code. The lysine residues that were replaced by aspartates are displayed in blue with chymotrypsinogen numbering above the bar. (B) GzmB variants were incubated with HL-60 cells at 4°C and detected by cell surface staining with the PE-labeled anti-GzmB antibody GB11-PE and analyzed by flow cytometry (SI Materials and Methods). (C) HL-60 cells were preincubated with unlabeled competitor proteins, and then GzmB-bio (10 μg/ml) was added at 4°C. Binding of GzmB-Bio was analyzed by flow cytometry using SA-PE (SI Materials and Methods). Shown are normalized data pooled from several independent experiments with 100% representing geometrical mean fluorescence intensity (MFI) without competitor proteins [GzmB, GzmB^FacD, and GzmB^KD-FacD, n = 3; GzmB^KD, n = 1; GzmB-CD₈, n = 2 (all ± SD)]. (D) Confocal imaging of HL-60 cells with endocytosed GzmB-Alexa⁶³³ or GzmB^KD-FacD-Alexa⁶³³ after 1 h of incubation at 37°C (SI Materials and Methods). Blue, Hoechst 33342; red, Alexa Fluor 633-labeled granzymes. (Scale bar: 20 μm.) (E) Quantification of endocytosed granzymes 1 h after incubation at 37°C by intracellular FACS staining with GB11-PE. (Left and Center) Histograms of stainings at the indicated granzyme concentrations. (Right) Summary of two independent, pooled experiments (background subtracted).

Fig. 2.

Decreased inhibition of GzmB-mutants by heparin. (A) Heparin inhibits GzmB-mediated cell death. GzmB mutants (10 μg/ml) and heparin were added to HL-60 cells. Streptolysin O (SLO), at sublytic concentration, was applied to deliver granzymes. Two hours later, cell death and apoptosis were monitored by annexin V-FITC (AV)/propidium iodide (PI) staining. Dead (AV⁺/PI⁺) and apoptotic (AV⁺/PI⁻) cells were summed up. The columns and error bars indicate the mean of two independent experiments (n = 2 ± SD). (B) Apoptotic effects are abrogated by heparin because of direct inhibition of enzymatic activity. Granzymes (200 nM) and heparin were preincubated, and the activity was measured against Ac-IEPD-pNA. Reduction of the slope (initial increase in OD₄₀₅) was calculated as percentage (n = 2 ± SD) (SI Materials and Methods).

Fig. 3.

Apoptotic potential of GzmB mutants. GzmB mutants were delivered to the cytosol of HL-60 cells either by SLO (A) or by soluble pfn (B). (A) SLO transfers each mutant with similar efficacy (n = 2 ± SEM). (B) The pfn-induced transfer of GzmB variants is slightly impaired compared with WT-GzmB (n = 3 ± SEM).

Fig. 4.

Externally added GzmB and GzmB mutants complement NKL cells lacking GzmB. (A) Granzymes added externally to the medium correct the GzmB deficiency of NKL cells. K562 cells (Left) or HL-60 cells (Right) were coincubated with NKL cells. Apoptosis induced by NKL cells and monitored via [³H]thymidine release was very weak. Addition of GzmB or GzmB mutants (400 nM with K562 and 200 nM with HL-60 cells) strongly induced apoptosis even at very low effector-to-target (E:T) ratios. n = 2 ± SEM (Left), and n = 2–3 ± SEM (Right). (B) Dose-dependent apoptosis induction by GzmB and GzmB mutants. Granzymes were titrated over a wide concentration range for a given E:T ratio of 10:1 (NKL:HL60). n = 2 ± SEM. (C) Complementation effects are caspase- and pfn-dependent. Treatment with the pan-caspase inhibitor Z-VAD-FMK or concanamycin A (CMA) totally abolished [³H]thymidine release induced by granzymes (400 nM) and NK cells at an E:T ratio of 10:1. (n = 2 ± SEM).

Fig. 5.

Size dependence of GzmB entry. (A) Comparisons between the GzmB-MOG fusion and WT-GzmB show almost similar apoptotic activities in HL-60 cells after permeabilization with SLO (n = 2 ± SEM). (B) pfn-induced delivery of GzmB-MOG is moderately reduced (n = 3 ± SEM). (C) Delivery of GzmB-MOG by NKL-derived pfn was lower than that of WT-GzmB (E:T, 10:1) (n = 3 ± SEM). *, P < 0.05. (D) The mAb to MOG (MAB2439, 266 nM) is a rat IgG2b and forms a stable complex with GzmB-MOG (100 nM), which is pulled down by protein G-coupled Sepharose. The residual activity in the supernatant (ΔOD per hour) is depicted by columns, and the activity in the pellets after 1 h of incubation in substrate is shown by symbols. (E) pfn-mediated delivery is size-restricted. Granzymes at 200 nM, free or precomplexed with anti-MOG or its isotype (both 400 nM), were delivered to HL-60 cells by sublytic amounts of purified pfn (n = 2 ± SEM). (F) NKL cell-mediated delivery of exogenously added GzmB-MOG is specifically blocked by anti-MOG mAb. Same experimental setup as in E except that NKL cells were used instead of pfn. Release of [³H]thymidine by apoptotic HL-60 cells at an E:T of 10:1 was measured (n = 2 ± SEM). N.D., not determined; **, highly significant at the P < 0.0001 level; ns, not significant with P > 0.05.