TABLE 1.

Properties of GrB mutants^a

Granzyme	Heparin bindingb	Activityc	Cytotoxicityd		Binding/accumulatione		Intracellular localizationf
			SLO	Pfp			IGD	Cytoplasm
					− HeS	+ HeS	− HeS	+ HeS	− HeS	+ HeS
wt	1.00	1.0	1.0	1.00	1.00	0.38	+++	+	+	+
cs1	0.03	1.0	1.0	0.05	0.38	0.35	−	−	+	+
cs2	0.1	1.0	1.0	0.5	0.63	0.57	++	+/−	+	+
cs1+2	0.03	1.0	1.0	0.02	0.22	NDg	−	−	+	+

^aResults are normalized to wt GrB. Binding and localization of FITC-GrB was assessed in the presence of sublytic perforin for 15 min.

^bAssessed by heparin-Sepharose pull-down assay and densitometry (Fig. 4).

^cEnzymatic activity measured by hydrolysis of a synthetic peptide substrate (Fig. 4).

^dTarget cell death measured by MTT assay following granzyme delivery by streptolysin O (SLO) (Fig. 4) or perforin (Pfp) (Fig. 5).

^eBinding and accumulation of FITC-GrB was assessed by FACS analysis, in the absence (−) or presence (+) of heparin (HeS). Averaged mean fluorescence intensities (MFI) were normalized to the averaged MFI for wt GrB without heparin. The MFI for wt GrB was 12.5 ± 1.9 (n = 6).

^fIntracellular localization of FITC-GrB was assessed by CLSM, in the absence or presence of heparin. For IGD: +++, multiple, large IGD in most cells; ++, fewer, smaller IGD in most cells; +, fewer, less intense IGD in many cells; +/−, rare, less intense IGD in occasional cells; −, no detectable IGD. For cytoplasm: +, diffuse cytoplasmic staining in most cells.

^gND, not done.