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

Department of Medicine, Evanston Northwestern Healthcare Research Institute, 2650 Ridge Avenue, Evanston, Illinois 60201, USA. s-raja@northwestern.edu
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 approximately 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) approximately 0.8 microm), 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 approximately 4-8 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.
Mesh Terms:
Apoptosis, Biophysical Phenomena, Biophysics, Biosensing Techniques, Biotinylation, Blotting, Western, Chondroitin Sulfates, Dose-Response Relationship, Drug, Electrophoresis, Agar Gel, Electrophoresis, Capillary, Granzymes, Humans, Hydrogen-Ion Concentration, Killer Cells, Natural, Kinetics, Lasers, Light, Protein Binding, Proteoglycans, Scattering, Radiation, Sepharose, Serine Endopeptidases, Software, Surface Plasmon Resonance, Time Factors, Ultracentrifugation, Vesicular Transport Proteins
J. Biol. Chem. Dec. 20, 2002; 277(51);49523-30 [PUBMED:12388539]
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