Interaction of chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent on pH.
Cytolytic T-lymphocytes (CTL) harbor cytoplasmic granules containing the lytic, pore-forming protein perforin, a family of serine proteases designated granzymes, and proteoglycans as major constituents. Growth of CTL lines in the presence of PNP-xyloside completely inhibited the glycosylation of the granule-associated chondroitin sulfate A type proteoglycans. Only short glycosaminoglycan molecules were detected. The absence of intact proteoglycans neither altered the sorting of the granule-associated proteins perforin or granzyme A nor influenced their secretion into the extracellular milieu upon T-cell receptor complex stimulation. With a weak base, the pH of the granules was determined to be acidic. At pH 5.2, granzyme A and perforin formed complexes with chondroitin sulfate A. At neutral pH, perforin and only a minor fraction of granzyme A dissociated from the proteoglycan. Upon secretion of the granule contents induced by immobilized anti-CD3 antibodies, most granzyme A molecules remained complexed with the chondroitin sulfate A glycosaminoglycans, even if synthesis of intact proteoglycans was inhibited. We suggest that granule-associated molecules complex with proteoglycans under the acidic conditions prevailing in the trans Golgi and cytolytic granules. A possible pH shift occurring during exocytosis would cause perforin, but only a minor fraction of granzyme A, to dissociate from the proteoglycans.
Masson
D
D
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Peters
P J
PJ
Geuze
H J
HJ
Borst
J
J
Tschopp
J
J
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002501:Centrifugation, Zonal; D002809:Chondroitin Sulfates / Q000378:metabolism*; D002850:Chromatography, Gel; D002999:Clone Cells; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011509:Proteoglycans / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D011509:Proteoglycans; D054353:Perforin; D002809:Chondroitin Sulfates; D012697:Serine Endopeptidases