Active Granulysin (GNLY)

Granulysin (GNLY) is a member of the saposin-like protein (SAPLIP) family of membrane disrupting proteins. Granulysin is expressed in granules of natural killer and activated cytotoxic T cells. It exhibits cytolytic activity against intracellular or extracellular microbes and also tumors, either alone or in synergy with perforin. Human granulysin has structural similarity and 30 - 40% aa identity to granulysins and NK-lysins of other mammals such as bovine, porcine and canine; similar peptides in rodents have not been identified. The 15 kDa unglycosylated protein contains five helical domains; helix 2 and 3 contain 9 arginines and one cysteine critical for activity. The activity of GNLY is usually measured by a cell apoptosis assay using Jurkat cells. Jurkat cells were seeded into triplicate wells of 96-well plates at a density of 8,000 cells/well with 5% serum standard 1640 which contains various concentrations of recombinant human GM-CSF. After incubated for 3 days, cells were observed by inverted microscope and cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Briefly, 10 µl of CCK-8 solution was added to each well of the plate, then the absorbance at 450 nm was measured using a microplate reader after incubating the plate for 2-4 hours at 37 ℃. Apoptosis of Jurkat cells after incubation with GNLY for 3 days observed by inverted microscope was shown in Figure 1. Cell viability was assessed by CCK-8 (Cell Counting Kit-8 ) assay after incubation with recombinant human GNLY for 3 days. The result was shown in Figure 2. It was obvious that GNLY significantly decreased cell viability of Jurkat cells. The ED50 is 0.84 ug/ml.