Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes.

Abstract

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.

Fluorogenic Granzyme A Substrates Enable Real‐Time Imaging of Adaptive Immune Cell Activity

“…We also tested their chemical integrity at different pH values because GzmA can be stored in slightly acidic cytotoxic granules inside CTLs. [21] In these experiments, we observed that the amide compound 4 was stable in basic and acid media, whereas the thioester 3 was more stable than the carboxylic ester analogue 2 at acidic pH (Figure 2 ). Finally, we confirmed by HPLC‐MS that the Ac‐IGNR amide derivative could be cleaved by recombinant hGzmA (Figure S1), thus we focused on this scaffold for the generation of fluorogenic GzmA substrates.…”

“…As shown in Figure 2, we found that compounds 2 and 3 were completely hydrolyzed in Tris buffer (pH 7.4) whereas compound 4 was fully stable under the same experimental conditions (purity >95 %). We also tested their chemical integrity at different pH values because GzmA can be stored in slightly acidic cytotoxic granules inside CTLs [21] . In these experiments, we observed that the amide compound 4 was stable in basic and acid media, whereas the thioester 3 was more stable than the carboxylic ester analogue 2 at acidic pH (Figure 2).…”