Granzyme B delivery via perforin is restricted by size, but not by heparan sulfate-dependent endocytosis.
How granzymes gain entry into the cytosol of target cells during killer cell attack has been the subject of several studies in the past, but the effective delivery mechanism during target cell encounter has not been clarified. Here we show that granzyme B (GzmB) mutants lacking binding to negatively charged, essentially heparan-sulfate-containing membrane receptors are poorly endocytosed yet are delivered to the cytosol with efficacy similar to that of WT GzmB. In a cell-based system GzmB-deficient natural killer cells provided perforin (pfn) by natural polarized secretion and synergized with externally added GzmB. Whereas receptor (heparan sulfate)-dependent endocytosis was dispensable, delivery of larger cargo like that of GzmB fusion proteins and GzmB-antibody complexes was restricted by their size. Our data support the model in which granzymes are primarily translocated through repairable membrane pores of finite size and not by the disruption of endocytosed vesicles. We conclude that structurally related translocators, i.e., perforin and cholesterol-dependent cytolysins, deliver deathly cargo across host cell membranes in a similar manner.
Kurschus
Florian C
FC
Department of Neuroimmunology, Max Planck Institute of Neurobiology, Am Klopferspitz 18, D-82152 Planegg-Martinsried, Germany.
Fellows
Edward
E
Stegmann
Elisabeth
E
Jenne
Dieter E
DE
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects; D053804:Granzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000494:pharmacology; D006801:Humans; D007694:Killer Cells, Natural / Q000201:enzymology; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D054353:Perforin / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
D053804:Granzymes; D006497:Heparan Sulfate; D054353:Perforin
Curr Opin Immunol. 2007 Jun;19(3):339-47
17442557
J Biol Chem. 2002 Dec 27;277(52):50923-33
12384499
Immunity. 2005 Sep;23(3):249-62
16169498
Chem Biol. 2005 May;12(5):567-77
15911377
Mol Cell Biol. 2005 Sep;25(17):7854-67
16107729
Cell. 2005 Apr 22;121(2):247-56
15851031
J Immunol. 1996 May 15;156(10):3678-86
8621902
Biochim Biophys Acta. 1991 Mar 18;1063(1):137-46
2015253
Blood. 2005 Mar 1;105(5):2049-58
15528317
J Biol Chem. 2004 May 7;279(19):20200-10
14985351
Microbes Infect. 2007 Sep;9(11):1341-50
17890127
J Biol Chem. 1985 Aug 5;260(16):9069-72
3874868
Science. 1986 Jul 11;233(4760):184-90
2425429
Science. 2007 Sep 14;317(5844):1552-4
17872444
J Biol Chem. 2005 May 27;280(21):20752-61
15788411
J Cell Biol. 2003 Jan 20;160(2):155-6
12538637
J Cell Biochem. 2005 Jun 1;95(3):598-610
15791691
Curr Opin Immunol. 2003 Oct;15(5):522-7
14499260
Nat Rev Immunol. 2003 May;3(5):361-70
12766758
Mol Biol Cell. 2006 Feb;17(2):623-33
16280358
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3185-90
11248053
Curr Opin Immunol. 2007 Jun;19(3):301-8
17433871
Protein Sci. 1992 Apr;1(4):426-71
1304349
Nature. 1983 Mar 31-Apr 6;302(5907):442-5
6835377
Biol Chem. 2006 Jul;387(7):827-37
16913832
FEBS Lett. 2004 Mar 26;562(1-3):87-92
15044006
Cell Death Differ. 2006 Apr;13(4):576-85
16179940
J Immunol. 2005 May 1;174(9):5456-61
15843543
J Biol Chem. 1985 Nov 25;260(27):14802-9
4055801
J Biol Chem. 2005 Jun 24;280(25):23549-58
15843372
Mol Cancer Ther. 2003 Dec;2(12):1341-50
14707275
J Immunol. 1985 Dec;135(6):4245-51
2415607
Science. 2007 Sep 14;317(5844):1548-51
17717151
Nat Rev Immunol. 2006 Dec;6(12):940-52
17124515
Biophys J. 2000 Feb;78(2):719-30
10653785