Cytotoxic cell granule-mediated apoptosis - Characterization of the macromolecular complex of granzyme B with serglycin

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16,417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB-SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of similar to 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K-d similar to 0.8 pm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB-SG interaction. Finally, characterization of GrB-SG interactions within granules revealed complexes of two distinct molecular sizes, one held similar to4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.