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 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.
Raja
Srikumar M
SM
Department of Medicine, Evanston Northwestern Healthcare Research Institute, 2650 Ridge Avenue, Evanston, Illinois 60201, USA. s-raja@northwestern.edu
Wang
Baikun
B
Dantuluri
Mandakini
M
Desai
Umesh R
UR
Demeler
Borries
B
Spiegel
Katharina
K
Metkar
Sunil S
SS
Froelich
Christopher J
CJ
D017209:Apoptosis; D055592:Biophysical Phenomena; D001703:Biophysics; D015374:Biosensing Techniques; D019921:Biotinylation; D015153:Blotting, Western; D002809:Chondroitin Sulfates / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary; D053804:Granzymes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007694:Killer Cells, Natural / Q000166:cytology / Q000473:pathology*; D007700:Kinetics; D007834:Lasers; D008027:Light; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism / Q000494:pharmacology*; D012542:Scattering, Radiation; D012685:Sepharose / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000494:pharmacology*; D012984:Software; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014461:Ultracentrifugation; D033921:Vesicular Transport Proteins
D011509:Proteoglycans; D033921:Vesicular Transport Proteins; C075430:serglycin; D002809:Chondroitin Sulfates; D012685:Sepharose; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases