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Vol. 17, Issue 2, 623-633, February 2006

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Granule-mediated Killing by Granzyme B and Perforin Requires a Mannose 6-Phosphate Receptor and Is Augmented by Cell Surface Heparan Sulfate

Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Submitted July 14, 2005; Revised October 12, 2005; Accepted November 2, 2005
Monitoring Editor: Suzanne Pfeffer

During granule-mediated killing by cytotoxic T lymphocytes or natural killer cells, the serine protease granzyme B enters the target cell by endocytosis and induces apoptosis. Previous studies suggested a role for the mannose 6-phosphate receptor, but further experiments with purified granzyme B indicated this was not essential. Additionally, it is now clear that grB is exocytosed from killer cells in a high-molecular-weight complex with the proteoglycan serglycin. Here granzyme B was delivered as a purified monomer, or in complex with either glycosaminoglycans or serglycin, and killing was evaluated. When granzyme B was a monomer, soluble mannose 6-phosphate had a limited impact, whereas apoptosis induced by the complexed grB was effectively inhibited by mannose 6-phosphate. Most importantly, when granzyme B and perforin were delivered together from granules, inhibition by mannose 6-phosphate was also observed. In pulldown assays mediated by the cation-independent mannose 6-phosphate receptor, granzyme B bound to the receptor more intensely in the presence of immobilized heparan sulfate. We therefore propose the model that under physiological conditions serglycin-bound granzyme B is critically endocytosed by a mannose 6-phosphate receptor, and receptor binding is enhanced by cell surface heparan sulfate.

Abbreviations used: AdV, adenovirus; bCI-MPR, biotinylated CI-MPR; CI-MPR, cation-independent mannose 6-phosphate receptor; ConA, concanavalin A; CTL, cytotoxic T lymphocyte; GAG, glycosaminoglycan; grB, granzyme B; M6P, mannose 6-phosphate; MPR, mannose 6-phosphate receptor; NK, natural killer; SLO, streptolysin O; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

^* These authors contributed equally to this work.

Address correspondence to: Chris Bleackley (chris.bleackley{at}ualberta.ca).

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