Inactivation and proteolytic degradation of perforin within lytic granules upon neutralization of acidic pH

被引:29
作者
Kataoka, T [1 ]
Togashi, K [1 ]
Takayama, H [1 ]
Takaku, K [1 ]
Nagai, K [1 ]
机构
[1] MITSUBISHI KASEI INST LIFE SCI,CELLULAR IMMUNOL LAB,MACHIDA,TOKYO 194,JAPAN
关键词
D O I
10.1046/j.1365-2567.1997.00257.x
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
In our recent studies, an inhibitor of vacuolar-type H+-ATPase, concanamycin A (CMA) has been shown to neutralize acidic pH in vacuolar organelles, including lytic granules, and to decrease the perforin content markedly. In the present paper, we have further investigated the role of acidification in perforin storage by using CMA. In CD8(+) cytotoxic T-lymphocyte (CTL) clones, the amount of perforin decreased rapidly at 30-90 min but no more decrease occurred at 90-120 min after the addition of CMA. Since exposure to actinomycin D, cycloheximide, or brefeldin A failed to reduce the perforin content, the perforin decrease in CMA-treated cells seems to be largely due to a reduction in the perforin already stored in lytic granules, rather than to the inhibition of the de novo synthesis or the intracellular glycoprotein transport of perforin. Diisopropylfluorophosphoridate (DFP) markedly antagonized the decrease in the perforin content in CMA-treated cells, while other protease inhibitors, i.e. antipain, E-64, leupeptin, pepstatin A and phenylmethylsulphonyl fluoride, did not. Nevertheless, DFP hardly reversed the abrogation of the killing activity by CMA. Indeed, the lytic granules prepared from DFP plus CMA-treated cells showed only a marginal level of haemolytic activity. In cell-free experiments using perforin-enriched granule fractions, acidic pH completely blocked the perforin activity. Under the acidic conditions, perforin was more resistant to an inactivation by calcium when exposed to calcium prior to the haemolysis test. Thus, these data suggest that perforin is primarily inactivated, possibly in a calcium-dependent manner, and is subsequently hydrolysed by DFP-sensitive proteases in the lytic granules at neutral pH. We conclude that acidic pH plays an essential role to maintain the integrity of perforin within the lytic granules.
引用
收藏
页码:493 / 500
页数:8
相关论文
共 31 条
[1]  
BASHFORD CL, 1988, J IMMUNOL, V141, P3965
[2]   THE LYTIC GRANULES OF NATURAL-KILLER-CELLS ARE DUAL-FUNCTION ORGANELLES COMBINING SECRETORY AND PRE-LYSOSOMAL COMPARTMENTS [J].
BURKHARDT, JK ;
HESTER, S ;
LAPHAM, CK ;
ARGON, Y .
JOURNAL OF CELL BIOLOGY, 1990, 111 (06) :2327-2340
[3]  
CRIADO M, 1985, J IMMUNOL, V135, P4245
[4]   INHIBITORY EFFECT OF MODIFIED BAFILOMYCINS AND CONCANAMYCINS ON P-TYPE AND V-TYPE ADENOSINE-TRIPHOSPHATASES [J].
DROSE, S ;
BINDSEIL, KU ;
BOWMAN, EJ ;
SIEBERS, A ;
ZEECK, A ;
ALTENDORF, K .
BIOCHEMISTRY, 1993, 32 (15) :3902-3906
[5]   THE CALCIUM-BINDING PROTEIN CALRETICULIN IS A MAJOR CONSTITUENT OF LYTIC GRANULES IN CYTOLYTIC-T LYMPHOCYTES [J].
DUPUIS, M ;
SCHAERER, E ;
KRAUSE, KH ;
TSCHOPP, J .
JOURNAL OF EXPERIMENTAL MEDICINE, 1993, 177 (01) :1-7
[6]   GENERATION OF CONTINUOUS LARGE ANTIGRANULOCYTES LYMPHOCYTE LINES BY INTERLEUKIN-2 FROM THE SPLEEN-CELLS OF MICE INFECTED WITH MOLONEY LEUKEMIA-VIRUS - INVOLVEMENT OF INTERLEUKIN-3 [J].
HATTORI, M ;
SUDO, T ;
IIZUKA, M ;
KOBAYASHI, S ;
NISHIO, S ;
KANO, S ;
MINATO, N .
JOURNAL OF EXPERIMENTAL MEDICINE, 1987, 166 (04) :833-849
[7]   CYTOLYTIC ACTIVITY OF PURIFIED CYTOPLASMIC GRANULES FROM CYTO-TOXIC RAT LARGE GRANULAR LYMPHOCYTE TUMORS [J].
HENKART, PA ;
MILLARD, PJ ;
REYNOLDS, CW ;
HENKART, MP .
JOURNAL OF EXPERIMENTAL MEDICINE, 1984, 160 (01) :75-93
[8]  
HUDIG D, 1991, J IMMUNOL, V147, P1360
[9]   PROTEASES AND LYMPHOCYTE CYTOTOXIC KILLING MECHANISMS [J].
HUDIG, D ;
EWOLDT, GR ;
WOODARD, SL .
CURRENT OPINION IN IMMUNOLOGY, 1993, 5 (01) :90-96
[10]   Identification of low molecular weight probes on perforin- and fas-based killing mediated by cytotoxic T lymphocytes [J].
Kataoka, T ;
Taniguchi, M ;
Yamada, A ;
Suzuki, H ;
Hamada, S ;
Magae, J ;
Nagai, K .
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 1996, 60 (10) :1726-1728