title: Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells.
abstract: Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 reduces accumulation in this intracellular compartment and cytotoxicity two- to threefold. We also show that GrB-mediated cytotoxicity is abrogated by heparin and that target cells deficient in cell surface sulfate or glycosaminoglycans resist GrB. However, heparin does not completely prevent GrB internalization and chondroitin 4-sulfate does not inhibit cytotoxicity, suggesting that glycosaminoglycans are not essential GrB receptors. We propose that GrB enters cells by nonselective adsorptive pinocytosis, exchanging from chondroitin sulfate on serglycin to anionic components of the cell surface. In this electrostatic "exchange-adsorption" model, cs1 and cs2 participate in binding of GrB to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.
authors.forename: Catherina H
authors.initials: CH
authors.lastname: Bird
authors.forename: Jiuru
authors.initials: J
authors.lastname: Sun
authors.forename: Kheng
authors.initials: K
authors.lastname: Ung
authors.forename: Diana
authors.initials: D
authors.lastname: Karambalis
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Trapani
authors.forename: Phillip I
authors.initials: PI
authors.lastname: Bird
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D002384:Catalysis; D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D015683:Gene Products, gag / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D053804:Granzymes; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008360:Mannosephosphates / Q000737:chemistry / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D055672:Static Electricity
chemical_list: D002412:Cations; D015683:Gene Products, gag; D006025:Glycosaminoglycans; D008360:Mannosephosphates; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; C027693:mannose-6-phosphate; D006493:Heparin; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases