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Molecular and Cellular Biology, September 2005, p. 7854-7867, Vol. 25, No. 17
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.17.7854-7867.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cationic Sites on Granzyme B Contribute to Cytotoxicity by Promoting Its Uptake into Target Cells

Catherina H. Bird,1 Jiuru Sun,1 Kheng Ung,1 Diana Karambalis,1 James C. Whisstock,1 Joseph A. Trapani,2 and Phillip I. Bird1*

Department of Biochemistry and Molecular Biology, Monash University, Melbourne 3800, Australia,1 Cancer Immunology Program, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St., Melbourne 8006, Australia2

Received 21 April 2005/ Returned for modification 5 May 2005/ Accepted 17 June 2005

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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Building 13D, Room D320, Monash University, Melbourne 3800, Australia. Phone: 61 3 9905 3771. Fax: 61 3 9905 3726. E-mail: phil.bird{at}med.monash.edu.au.

Molecular and Cellular Biology, September 2005, p. 7854-7867, Vol. 25, No. 17
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.17.7854-7867.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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