Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line.
Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1ng/mL of granzyme B, compared to 1.5-2.5 microg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.
Pulmonary Research Group, Department of Medicine, University of Alberta, 550A Heritage Medical Research Centre, Edmonton, Alta., Canada.
Marcelo
M
Marcet-Palacios
Solomon O
SO
Odemuyiwa
Jason J
JJ
Coughlin
Daniella
D
Garofoli
Catherine
C
Ewen
Courtney E
CE
Davidson
Mazyar
M
Ghaffari
Kevin P
KP
Kane
Paige
P
Lacy
Michael R
MR
Logan
A Dean
AD
Befus
R Chris
RC
Bleackley
Redwan
R
Moqbel
D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000378:metabolism*; D050683:R-SNARE Proteins / Q000378:metabolism*; D050600:SNARE Proteins / Q000378:metabolism*
D050683:R-SNARE Proteins; D050600:SNARE Proteins; C121241:VAMP7 protein, human; D053804:Granzymes