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The Journal of Immunology, 1998, 160: 4988-4993.
Copyright © 1998 by The American Association of Immunologists

Purification and Characterization of Lymphocyte Chymase I, a Granzyme Implicated in Perforin-Mediated Lysis1

Susan L. Woodard*, Stephanie A. Fraser{ddagger}, Ulrike Winkler*, Delwin S. Jackson§, Chih-Min Kam§, James C. Powers§ and Dorothy Hudig2,*,{dagger}

* Department of Microbiology, School of Medicine, {dagger} School of Veterinary Sciences, and {ddagger} The Cell and Molecular Biology Graduate Program, University of Nevada, Reno, NV 89557; and § School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37°C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There are multiple pathways that cytotoxic lymphocytes utilize in the killing of target cells, including the Fas-ligand-Fas pathway (1) and the granule exocytosis pathway (2, 3; also reviewed in Ref. 4). The granule exocytosis pathway utilizes perforin and serine proteases of the granules (granzymes) to kill target cells. The tryptase granzymes A and K and the Asp-ase granzyme B mediate cytotoxic activity through their ability to induce DNA fragmentation after entering cells through perforin pores (5, 6, 7, 8, 9). Another granzyme, a chymase (with chymotrypsin-like specificity of cleavage), has a role in perforin pore formation (10, 11).

Lymphocyte chymase granzymes have been difficult to purify to date. The enzyme activity has been detected in cytotoxic rat (10) and human (M. Poe, unpublished results) lymphocytes. From computer models of granzyme serine protease active sites, mouse granzyme genes D, E, F and G, rat granzyme P7, and human granzyme H are predicted to be chymases (Refs. 12–14, respectively). To date, the only chymase purified from cytolytic granules has been a carboxypeptidase, cathepsin A-like protective protein (15). The abundance of the mRNA for granzymes D, E and F suggests that these granzymes may be expressed at ~100-fold lower concentrations than granzyme B (16), and, thus, enzyme recovery may be restricted by the initial low amounts.

We recently identified a chymase-directed serine protease inhibitor, Bi-Aca-Aca-Phe-Leu-PheP(OPh)2,3 that preferentially reacts with one granzyme of rat RNK-16 NK cells (17). This biotin-tagged reagent also prevented granule-mediated killing and blocked NK killing (17). Here we describe the purification and characterization of granzyme chymase I, the chymase that reacts preferentially with this tagged inhibitor. Our starting material was granule extracts from 6 to 30 x 109 rat RNK-16 lymphocytes per preparation of chymase I.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of chymase I

Chymase I was purified from RNK-16 cell granules by size exclusion and cation exchange chromatography.

Isolation of RNK-16 granules. RNK-16 cells (18) were grown as ascites cells in F344 rats. Our RNK-16 subline has retained granule lytic activity while the live cells no longer bind to or kill YAC-1 target cells. To obtain the granules, the cells were disrupted by nitrogen cavitation, and the lysate was centrifuged over 54% Percoll (Sigma Chemical, St. Louis, MO). Granules were isolated from the dense portion of the gradient as previously described (17). The granule pool was made 1 M in NaCl and then freeze thawed three times to disrupt the granule membranes. The granule extract was stored at -20°C. The yield was ~0.5 mg of granule extract per billion RNK-16 cells.

Superdex 200 size exclusion chromatography. Twenty milliliters of granule extract were concentrated approximately fivefold by centrifugation with Centriprep 10 concentrators (Amicon, Beverly, MA) before loading 3 ml onto a 2.6 x 70-cm Superdex 200 (Pharmacia Fine Chemicals, Piscaway, NJ) column. The running buffer was 20 mM HEPES, 1 M NaCl, 10% betaine, 0.1 mM EGTA, and 0.05% NaN3. Aprotinin (6.5 kDa), carbonic anhydrase (29 kDa), BSA (66 kDa), and IgG (150 kDa) were used for size calibration. The void volume was determined using blue dextran (2000 kDa).

MonoS cation exchange chromatography. The leading one-third of the SD200 low m.w. chymase peak was omitted from the pool loaded onto an HR5/5 MonoS fast protein liquid chromatography (FPLC) column (Pharmacia) because these fractions contained granzyme B that would copurify with chymase I on MonoS. The SD200 chymase pool was diluted with an equal volume of NaCl-free buffer (125 mM MES, 0.15 mM EGTA, 15% betaine, pH 5.9). Pure ethylene glycol (enzyme grade, Fisher Biotech, Fair Lawn, NJ) was added to make the chymase solution 0.4 M NaCl, 20% ethylene glycol, 50 mM MES, 10% betaine, 0.05% NaN3, and 0.1 mM EGTA, pH 6.0. The diluted sample was then in MonoS running buffer A. MonoS running buffer B was similar to buffer A but with 1 M NaCl. The sample was loaded and washed in 0.4 M NaCl (60% A, 40% B). A linear gradient was then run from 40% to 100% B. In additional experiments not illustrated, chymase I was isolated using immobilized metal affinity chromatography (IMAC) with Cu2+ bound to Poros IMAC beads (PerSeptive Biosystems, Cambridge, MA) (19).

Chymase detection

Substrate hydrolysis. Rates of hydrolysis of peptide thiobenzyl substrates were measured using Ellman’s reagent, dithiobis-(2-nitrobenzoic acid) (1.2 mM in assay), to monitor the production of HSBzl. The extinction coefficient for the colored thiol product at 410 nm is 13,600 M-1 cm-1 (20). Rates were measured in esterase buffer (0.1 M HEPES, 0.5 M NaCl, pH 7.5) at 25°C. For assaying activity from column runs, 10 to 100 µl of each fraction were assayed in a total volume of 200 µl in 96-well microtiter plates using a Molecular Devices kinetics microplate reader (Palo Alto, CA). Rates were determined in duplicate and averaged. The substrate Suc-Phe-Leu-Phe-SBzl (Bachem Bioscience, King of Prussia, PA) was used for the chymase assays, except where indicated in Table IIGo. Boc-Lys-SBzl (Sigma), Boc-Ala-Ala-Met-SBzl (Enzyme System Products (ESP), Dublin, CA), and Boc-Ala-Ala-Asp-SBzl (ESP) were used to measure tryptase, Met-ase, and Asp-ase activities, respectively. The Boc-Ala-Ala-[P1 = Leu, Phe, Ser, Trp, or Tyr]-SBzl substrates were synthesized in the laboratory of J.C.P. (21). Updated procedures for synthesis are available upon request. Substrates were used at 90 µM, except for Suc-Phe-Leu-Phe-SBzl, for which solubility limited its concentration to 28 µM in the assay.


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  		Table II. Chymase I hydrolysis of peptide thiobenzyl ester substrates

 
Biotinylation with Bi-Aca-Aca-Phe-Leu-PheP(OPh)2. Chymase-containing fractions were pretreated with either 0.1 mM inhibitor Bi-Aca-Aca-Phe-Leu-PheP(OPh)2 (22) or the solvent DMSO for 10 min at room temperature. The treated fractions were assayed for loss of enzyme activity and for biotinylation using protein blots (17).

Protein characterization

Protein determinations. Protein assays were done with bicinchoninic acid (BCA) (23) (Pierce, Rockford, IL) with crystalline BSA as a standard.

Gel electrophoresis. Samples were run on SDS-PAGE under reducing conditions using 12% MiniProtean gels (Bio-Rad Laboratories, Richmond, CA). Proteins were detected by silver staining.

Protein sequencing. Protein sequencing was performed on an Applied Biosystems (Foster City, CA) Procise 494 gas phase sequencer by Dr. Matthew Williamson (University of California at San Diego, San Diego, CA). The protein was bound to ProBlott (Applied Biosystems) polyvinylidene difluoride (PVDF) membrane for sequencing.

Determination of pH optimum

Hydrolysis rates of Suc-Phe-Leu-Phe-SBzl were measured in Good’s buffer solutions containing 0.1 M MES (pH 5.58, 5.87, 6.12, and 6.43), 0.1 M PIPES (pH 6.44, 6.83, and 7.23), 0.1 M HEPES (pH 7.09, 7.50, 7.75, and 7.94) or 0.1 M TAPS (pH 7.94, 8.33, 8.69, and 9.10) and 0.75 M NaCl in the presence of 1.2 mM Ellman’s reagent. The data were fit using a nonlinear least squares algorithm to an equation of the form y = ax2 + bx + c.

Chymase inhibition

Chymase isolated after SD200 was treated with 0.1-mM inhibitors for 20 min at room temperature in esterase buffer and then immediately assayed. The inhibitors were dissolved at 20 to 40 mM in anhydrous DMSO and stored at -20°C. Controls were treated with DMSO alone. The inhibitors came from the following sources: 3,4-dichloroisocoumarin (Boehringer Mannheim, Indianapolis, IN); PMSF and Tosyl-Phe-CH2Cl (Sigma); Z-Gly-Leu-Phe-CH2Cl (ESP). 2-(Z-NH(CH2)2CO-NH)C6H4SO2F (24), Bi-Aca-Aca-Phe-Leu-PheP(OPh)2 (22), Suc-Phe-Leu-PheP(OPh)2, (25) and FITC-Ala-Ala-MetP(OPh)2 (25) were synthesized in the laboratory of J.C.P.

Chymase effects on perforin lysis

Purified chymase I (or other granzymes) and isolated perforin were incubated together at 37°C for 30 min and then diluted before hemolytic assays. Perforin was prepared by Cu2+-IMAC (Poros resin; PerSeptive Biosystems) (19), followed by phenyl-Superose (Pharmacia) hydrophobic interaction chromatography (26). In detail, 50 ml of perforin solution was mixed with 100 ml of chymase I, dilutions of chymase I in MonoS buffer, or MonoS buffer as a control and incubated. Then 850 ml of salt-free 10 mM HEPES pH 7.5 buffer was added (to bring the solution to physiologic salt), and the solution was diluted with HEPES-buffered saline (10 mM HEPES, pH 7.5, 0.15 M NaCl) with 10 µg/ml BSA (crystalline; U.S.Biologic, Cleveland, OH, No. 10856). In some experiments, the preincubation was omitted. Lysis was measured using five twofold serial dilutions of perforin (starting with a 1/20 final dilution) with rabbit RBC. The amount of perforin needed to lyse 50% of the erythrocytes was defined as 1 LU and calculated by linear regression of the log of the volume of perforin assayed vs lysis. Lysis of treated perforin was compared on the basis of LU per milliliter of perforin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chymase I purification

Chymase I, the granzyme preferentially reactive with Bi-Aca-Aca-Phe-Leu-PheP(OPh)2, was purified from RNK-16 granule extracts with approximately 30% recovery and 500-fold enrichment (Table IGo). The initial separation, by Superdex 200 size exclusion (Fig. 1GoA), was selected because the step was compatible with the high salt (>0.4 M NaCl) needed to stabilize the chymase activity. The high salt was also needed to separate the chymase from the high Mr proteoglycan (27) that complexes with granzymes (28) and interferes with their purification. Size exclusion also depleted most of the tryptases (GrA and GrK) and the Asp-ase (GrB) from the chymase peaks (Fig. 1GoB). The majority of granule protein (as indicated by the OD280) eluted with the void volume where the high Mr proteoglycan also eluted (not indicated). There were four chymase peaks with apparent molecular masses of 600, 190, 88, and 14 kDa. The peak with the lowest apparent Mr was the most reactive with the biotinylated inhibitor (>99% inactivation after treatment with 0.1 mM for 10 min) and remained inhibited after dialysis. The first peak (600 kDa) was 28% inhibited and the second (190 kDa) peak of chymase was ~68% inhibited when 50 µM inhibitor was present in the enzyme assays. The third peak of chymase was unaffected. Neither the first or second chymase peak was inhibited after dialysis (or labeled with biotin detectable by SDS-PAGE). These data are consistent with unfavorable positioning of the inhibitors’s reactive phosphonate with these chymases, despite ability of the peptide inhibitor to compete with the peptide thioester substrate in the assays.


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  		Table I. Purification of chymase I

 


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  		FIGURE 1. Separation of chymase I by Superdex 200 size exclusion chromatography. Three milliliters of concentrated granule extract was loaded onto the Superdex 200 column. A, OD280 and chymase activities. B, Asp-ase (GrB), tryptase (GrA the larger, and GrK the smaller, tryptase), and Met-ase activities.

 
The net recovery of total chymase after sizing was close to the initial activity. From this recovery, we assumed that the initial amount of chymase I in the granules was proportional to the ~10% fraction appearing in the lowest Mr peak. We used the activity of this peak as 100% for our determination of the relative enrichment and recovery of chymase I (Table IGo). MonoS cation exchange chromatography was used as the second step of purification. The ion exchange depleted the contaminant tryptase GrK from the chymase (Fig. 2Go). The leading SD200 fractions of chymase I were excluded from further purification because they contained Asp-ase, GrB, which would copurify with chymase I by MonoS chromatography (not illustrated). Chymase I eluted from MonoS at ~650 mM NaCl. The overall enzymatic yield of chymase I after purification was ~30%. The protein yield was 8 µg from 13.7 mg of starting protein. This recovery indicates that the maximal abundance of chymase I protein within the granule extract is less than 0.2% of the total protein, based on calculations assuming 30% recovery of the protein (rather than better than 30% recovery, which could be the case if some of the enzyme lost activity).



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  		FIGURE 2. Final purification of chymase I by MonoS cation exchange chromatography. The low Mr chymase peak separated by SD200 chromatography was diluted to reduce the salt concentration and to adjust the buffer pH before ion exchange (see Materials and Methods). The leading third of this chymase peak was omitted from the pool that was loaded onto the MonoS column to eliminate GrB (Asp-ase).

 
Purified chymase I appears homogeneous after SDS PAGE. It was detected as a single silver-stained protein (Fig. 3GoA) and as a single biotinylated protein after reaction with the selective inhibitor (Fig. 3GoB). Chymase I has a molecular mass of ~30 kDa by SDS PAGE (Fig. 3Go), which indicated that it was nonspecifically retained on the SD200 column (where it eluted with 15-kDa proteins, Fig. 3GoA, lane 2). Purified chymase from as many as 3 x 1010 RNK-16 cells was sequenced. The limited N-terminal amino acid sequence that we obtained was Ile-(Ile/Leu)-Gly and is similar to the N-terminal Ile-Ile-Gly-Gly sequence common among the granzymes (29). In addition, the chymases eluted with GrB, GrA, GrK, and the other granzymes in a single sharp peak when a Cu2+-IMAC column with an imidazole gradient was used to separate granzymes from other granule proteins (19). Chymase I could be separated from the other granzymes afterward by SD200 chromatography. However, the chymase recovery was lower than with the procedure of Table IGo.



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  		FIGURE 3. Characterization of the Mr and inhibitor-reactivity of chymase I by SDS-PAGE and protein blots. A, Purification of chymase I. Chymase I at different steps of purification was characterized by SDS-PAGE and silver stained in the gels. Lane 1, 1 µg of unfractionated granule extract; lane 2, 1 µg of SD200 low Mr chymase; and lane 3, 0.25 µg of MonoS chymase I. Lanes 1 and 2 were overdeveloped in the silver stains in an attempt to visualize the 31-kDa chymase I, which is invisible within the unfractionated granule extract and barely visible in the SD200 peak. B, Reactivity of chymase I and a chymase copurifying with perforin with Bi-Aca-Aca-Phe-Leu-PheP(OPh)2. After separation by SDS-PAGE, the proteins were blotted. The biotinylated protease inhibitor complexes were detected with avidin-alkaline phosphatase. Lane 1, Inhibitor-treated SD200 perforin lytic peak; lane 2, DMSO-treated SD200 perforin lytic peak; lane 3, Inhibitor-treated SD200 low m.w. chymase I; and lane 4, DMSO-control-treated SD200 low m.w. chymase peak. The numbers indicate the molecular mass of the markers in kDa.

 
Enzymatic properties of chymase I

Chymase I has a broad pH optimum between pH 7.5 and 9 (Fig. 4Go), which is the pH optimum characteristic of serine-dependent proteases. When the optimal pH was determined for unfractionated chymases within the granule extract, the maximum activity toward the same substrate was at pH 7.5 (data not shown), indicating that chymase I (optimal at pH 8.7) is different from the predominant chymases. Chymase I demonstrated a marked preference for tryptophan at the P1 amino acid of substrates (see Ref. 30 for substrate nomenclature) when used to hydrolyse several Boc-Ala-Ala-[P1=X]-SBzl substrates (Table IIGo). Comparisons were made at 90 mM substrate concentration. Ninety micromolar concentrations of substrate were employed to facilitate detection of the low hydrolysis rates of Leu and Ser P1 substrates. Hydrolysis of the Trp P1 substrate was more than twofold faster than hydrolysis of the Phe P1 substrate. It is noteworthy that there was no detectable hydrolysis of the Tyr P1 substrate by chymase I while this substrate was concurrently hydrolysed by the unfractionated granule extract. Km values were not determined due to the low availability of chymase I. The turnover rate for the Suc-Phe-Leu-Phe-SBzl substrate at the solubility limit of 28 µM was 4.5 pmols/s whereas the rate for Boc-Ala-Ala-Trp-SBzl at 90 µM was 7.5 pmols/s, both with 97 ng of chymase I, indicating that the commercial reagent was a reasonably sensitive substrate despite its lack of an optimal P1 amino acid. These substrates have also been used to indicate that chymase I is unique. Ratios of enzyme activities of this peak toward Suc-Phe-Leu-Phe-SBzl vs Boc-Ala-Ala-Tyr-SBzl substrates indicate that it (chymase I) is distinct; the other chymases hydrolysed Boc-Ala-Ala-Tyr-SBzl (not indicated).



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  		FIGURE 4. Determination of the pH optima for chymase I activity. Chymase I fractionated by size exclusion was assayed with a series of Good’s buffers at the pH indicated.

 
Chymase I was reactive with selected irreversible inhibitors including the general irreversible serine protease inhibitors, 3,4-dichloroisocoumarin (DCI) and PMSF (Table IIIGo). Chymase I was also inactivated by several irreversible chymase-selective inhibitors representing classes of compounds that preferentially react with and inactivate serine proteases, including sulfonyl fluorides, peptide chloromethyl ketones, and peptide phosphonates (Table IIIGo).


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  		Table III. Inhibition of chymase I1

 
Biologic properties of chymase I

A biotinylated serine protease inhibitor, Bi-Aca-Aca-Phe-Leu-PheP(OPh)2, inactivates perforin-mediated lysis (17). A chymase reactive with this inhibitor copurified with perforin on SD200. It was detectable when the ~70-kDa perforin fraction was treated with the inhibitor and then highly concentrated before SDS-PAGE and protein blotting. (See the biotinylated 30-kDa protein of Fig. 3GoB, lane 1). Purified chymase I also reacted with the biotinylated inhibitor (Fig. 3GoB, lane 3) and has the same Mr as the perforin-associated chymase. An additional ~45-kDa band visible in Figure 3GoB, lanes 1 and 2, is an endogenous avidin-binding protein found with the granule proteins. So far we have been unable to purify perforin to homogeneity and retain lytic activity. For this reason, we supplemented highly enriched (and still lytic) perforin with purified chymase I to determine its effects on perforin lysis. When perforin was preincubated with chymase I at 37°C (without calcium), there was a dose-dependent loss of lytic activity (Fig. 5Go). Without the preincubation, there was no effect on perforin lysis when identical amounts of chymase I were added in similar protocols. Because calcium will inactivate perforin during preincubation, there are two other differences in these protocols: the presence or absence of calcium and the availability of a membrane to accept the pore. Pretreatment of perforin with GrK or the two other chymase peaks separated by SD200 (at similar mOD min-1 ml-1 of enzyme activity) was without effect on lysis. Addition of human neutrophil cathepsin G, another "chymase," was also without effect. It should be noted that these preparations of perforin contain ~1 mOD min-1 of chymase per 1000 lytic units of perforin (~100 mOD min-1 per mg of perforin) (31).



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  		FIGURE 5. Supplementation of perforin with chymase I. Highly enriched perforin was incubated with the indicated concentrations of purified chymase I for 30 min at 37°C, diluted, and then assayed using further twofold dilutions for lysis of RBC. Specifically, the indicated concentrations (mOD min-1 ml-1) of chymase I (or buffer control) was added to a final volume of 0.15 ml with purified perforin and incubated (see Materials and Methods) before 20- to 80-fold dilutions in the lytic assays. The perforin preparation contained traces of other proteins, including perforin enhancing protein (PEPr) (26) and calreticulin (Ref. 40 and our unpublished results).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have purified a new rat chymase. We believe that it is the first lymphocyte chymase granzyme purified and characterized enzymatically. Chymase I has many properties typical of a serine protease of the chymotrypsinogen subfamily. The molecular mass of 30 kDa suggests that there are 1 or 2 glycosylation sites on a chymotrypsin-like structure that lacks additional domains. (The kringle structures of many blood coagulation proteases are examples of additional domains.) The pH optimum is also typical. Chymase I was inhibited by both DCI (32) and PMSF, "general" serine protease inhibitors that react with almost all serine proteases. The limited N-terminal amino acid sequence and its elution from Cu2+-IMAC suggest that chymase I is likely to be a granzyme.

We need additional protein sequence information to match chymase I with the gene that encodes it. Amino acid sequences extending beyond amino acid 17 of chymase I will be needed. Mature, fully processed granzymes conserve the first four N-terminal amino acids. The granzyme pairs D & E and F & G have amino acids 5 to 8 in common so that this sequence could be ambiguous. The granzymes retain a conserved motif at the next eight amino acids, 9 to 16. We are also developing an alternate approach for granzyme identification by making granzyme-specific Abs to peptide representing amino acids 17 to about 30 of the mature granzymes. Rat granzyme C (also termed P4; 13 , rat granzyme D/G1 (P7; 13 , and rat granzyme J (P5; 33 genes have been cloned and are not yet matched with the proteins they encode. Based on computer models of the S1 substrate binding pockets, chymase I could correspond to GrD/G1 and is unlikely to be encoded by the GrC or GrJ genes.

The enzymatic properties of chymase I have several implications. The pH optimum between 7 and 9 indicates that the chymase would have very little activity at the intragranular pH of 5.5 (28) and would need to be exocytosed to be fully active. Rat mast cell protease I, a chymase, also has optimal activity at pH 8.5 (34). The selective reactivity for tryptophan at P1 contrasts with the P1 specificity of the other rat lymphocyte chymases (S.A.F., unpublished results) and with the specificity of rat mast cell chymases I and II, both of which have much greater activity toward Phe than Trp at P1 as reflected in kcat/Km that are 209- and 90-fold better for Phe than Trp in Suc-Val-Pro-(Phe/Trp)-NA substrates (24). This reactivity suggests that lymphocyte chymase I-selective inhibitors could be designed that would incorporate the P1 amino acid Trp. The chymase-directed sulfonyl fluoride inhibitor 2-(Z-NH(CH2)2CO-NH)C6H4SO2F, the Z-Gly-Leu-Phe-CH2Cl chloromethyl ketone inhibitor, and the peptide phosphonate inhibitors that inactivated chymase I also blocked perforin-mediated lysis (see Refs. 10, 35, and 17, respectively.)

Several observations are consistent with a physiologic role for chymase I although additional experiments will be needed to establish this role. 1) Chymase I reacts preferentially with Bi-Aca-Aca-Phe-Leu-PheP(OPh)2, an irreversible peptide phosphonate inhibitor that biotinylated one granule protein (Fig. 7, 17 , inactivated granule-mediated (perforin) lysis of RBCs, and blocked rat NK activity (17). 2) A similar (if not identical) reactive chymase with an equivalent Mr was observed in lytic perforin preparations (Fig. 3Go). 3) Furthermore, treatment of the perforin preparations with the biotinylated inhibitor also blocked lytic activity (our unpublished results). A simple interpretation is that this 30-kDa chymase participates in perforin-mediated cytotoxicity. For this interpretation to be valid, chymase I would have to have a human species equivalent. Granzyme H (14, 36), so far found only in humans, is a potential species homologue for equivalent function because granzyme H contains a large, apparently uncharged, substrate-binding pocket that is able to accommodate aromatic amino acids. The other human granzymes (A, B, K, and M) lack chymase activity (J.C.P., unpublished data).

We also found that preincubation of enriched perforin with purified chymase I depressed lytic activity. The experimental conditions are compatible with premature proteolysis of a perforin-associated granule protein by chymase I. Under calcium- and membrane-free conditions, the nascent product may have assumed a nonfunctional and irreversible conformation that will not enhance lysis. Lysis was reduced as a consequence when calcium and red cells are provided. Because the perforin contains only residual chymases, the enzyme supplementation (to physiologic ratios) may be required to produce its effects during preincubation.

The reduction of lysis could be interpreted as counter to a positive regulatory role for chymase I in lysis. A down-regulatory role for chymase I would place it as an intragranule protein to limit lysis of "by-stander" cells that are not targeted for lysis and add chymase I to the plasma proteins protein S (vitronectin) (37) and apolipoprotein B (38, 39), which reduce perforin’s lytic activity. However, we suggest that the issue of up- vs down-regulation of lysis remains open. Chymase I definitely reacts with a protein important to lysis. This interaction appears specific because the other endogenous chymases lacked an effect on lysis under the same conditions.

The specificity of chymase I is also reflected by the enzymatic and the genetic variability among the chymase granzymes. Chymase I prefers Trp P1 while the two other rat lymphocyte chymases that are in separate SD200 peaks prefer Phe to Trp at P1 (S.A.F., unpublished results). The rodent granzymes predicted to encode chymases differ in the genetic regions that specify their substrate binding sites (12). At this time, perforin and perforin enhancing protein (PEPr; 26 are two proteins that participate in lysis that are potential natural substrates to examine for selective hydrolysis by lymphocyte chymase I.

In summary, we have purified and characterized a native lymphocyte chymase. Our data suggest that it (chymase I) may have a role in perforin lysis. At present, its absolute requirement for lysis is unresolved. The low abundance of this granzyme within granules indicates that recombinant expression of the rat chymase granzymes would greatly benefit future studies.


   Acknowledgments
 
We thank Drs. Kathleen Schegg and David Schooley of Biochemistry at the University of Nevada (Reno, NV) for helpful advice on protein isolation and sequencing, Dr. Matthew Williamson at the University of California at San Diego (San Diego, CA) for protein sequencing, and Dr. Thomas F. Kozel at the University of Nevada (Reno, NV) for helpful suggestions with the manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants CA38942 (D.H.), GM42212 (D.H. and J.C.P.), GM 54401 (J.C.P. and D.H.), and T32CA09563 (S.A.F. and U.W.). Back

2 Address correspondence and reprint requests to Dr. Dorothy Hudig, Mail Code 320, School of Medicine, University of Nevada, Reno, NV 89557. E-mail address: Back

3 Abbreviations used in this paper: P(OPh)2, phosphonate phenylester; Aca, 6-aminocaproic acid; Bi, biotinyl; Boc, butyloxycarbonyl; IMAC, immobilized metal affinity chromatography; MES,2-morpholinoethanesulfonic acid; RNK, rat NK cell; SBzl, thiobenzyl; Suc, succinyl; Z, benzyloxycarbonyl; DCI, 3,4-dichloroisocoumarin. Back

Received for publication July 28, 1997. Accepted for publication January 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nagata, S., P. Golstein. 1995. The Fas death factor. Science 267:1449.[Abstract/Free Full Text]
  2. Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner, P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265:528.[Abstract/Free Full Text]
  3. Lowin, B., M. Hahne, C. Mattmann, J. Tschopp. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370:650.[Medline]
  4. Podack, E. R.. 1995. Execution and suicide: cytotoxic lymphocytes enforce Draconian laws through separate molecular pathways. Curr. Opin. Immunol. 7:11.[Medline]
  5. Shi, L., R. P. Kraut, R. Aebersold, A. H. Greenberg. 1992. A natural killer cell granule protein that induces DNA fragmentation and apoptosis. J. Exp. Med. 175:553.[Abstract/Free Full Text]
  6. Shiver, J. W., L. Su, P. A. Henkart. 1992. Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A. Cell 71:315.[Medline]
  7. Shi, L., C. Kam, J. C. Powers, R. Aebersold, A. H. Greenberg. 1992. Purification of three lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target interactions. J. Exp. Med. 176:1521.[Abstract/Free Full Text]
  8. Heusel, J. W., R. L. Wesselschmidt, S. Shresta, J. H. Russell, T. J. Ley. 1994. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977.[Medline]
  9. Smyth, M. J., J. A. Trapani. 1995. Granzymes: exogenous proteinases that induce target cell apoptosis. Immunol. Today 16:202.[Medline]
  10. Ewoldt, G. R., U. Winkler, J. C. Powers, D. Hudig. 1992. Sulfonyl fluoride serine protease inhibitors inactivate RNK-16 lymphocyte granule proteases and reduce lysis by granule extracts and perforin. Mol. Immunol. 29:713.[Medline]
  11. Talento, A., M. Nguyen, S. Law, J. K. Wu, M. Poe, J. T. Blake, M. Patel, T. Wu, C. Manyak, M. Silberklang, G. Mark, M. Springer, N. H. Sigal, I. L. Weissman, R. C. Bleackley, E. R. Podack, M. L. Tykocinski, G. C. Koo. 1993. Transfection of mouse CTL with an antisense granzyme A vector reduces lytic activity. J. Immunol. 149:4009.[Abstract]
  12. Odake, S., C. Kam, L. Narasimhan, M. Poe, J. T. Blake, O. Krahenbuhl, J. Tschopp, J. C. Powers. 1991. Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry 30:2217.[Medline]
  13. Ewoldt, G. R., M. J. Smyth, P. K. Darcy, J. L. Harris, C. S. Craik, B. Horowitz, S. L. Woodard, J. C. Powers, D. Hudig. 1997. RNKP-4 and RNKP-7, new granzyme serine proteases expressed in activated rat lymphocytes. J. Immunol. 158:4574.[Abstract]
  14. Meier, M., P. C. Kwong, C. J. Fregeau, E. A. Atkinson, M. Burrington, N. Ehrman, O. Sorensen, C. C. Lin, J. Wilkins, R. C. Bleackley. 1990. Cloning of a gene that encodes a new member of the human cytotoxic cell protease family. Biochemistry 29:4042.[Medline]
  15. Hanna, W. L., J. M. Turbov, H. L. Jackman, F. Tan, C. J. Froelich. 1994. Dominant chymotrypsin-like esterase activity in human lymphocyte granules is mediated by the serine carboxypeptidase called cathepsin A-like protective protein. J. Immunol. 153:4663.[Abstract]
  16. Prendergast, J. A., C. D. Helgason, R. C. Bleackley. 1992. Quantitative polymerase chain reaction analysis of cytotoxic cell proteinase gene transcripts in T cells. J. Biol. Chem. 267:5090.[Abstract/Free Full Text]
  17. Woodard, S. L., D. S. Jackson, A. S. Abuelyaman, J. C. Powers, U. Winkler, D. Hudig. 1994. Chymase-directed serine protease inhibitor that reacts with a single 30-kDa granzyme and blocks NK-mediated cytotoxicity. J. Immunol. 153:5016.[Abstract]
  18. Ward, J. M., C. W. Reynolds. 1983. Large granular lymphocyte leukemia: a heterogeneous lymphocytic leukemia in F344 rats. Am. J. Pathol. 111:1.[Abstract]
  19. Winkler, U., T. M. Pickett, D. Hudig. 1996. Fractionation of perforin and granzymes by immobilized metal affinity chromatography (IMAC). J. Immunol. Methods 191:11.[Medline]
  20. Ellman, G. L.. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70.[Medline]
  21. Harper, J. W., R. R. Cook, C. J. Roberts, B. J. McLaughlin, J. C. Powers. 1984. Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II: bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates. Biochemistry 23:2995.[Medline]
  22. Abuelyaman, A. S., D. S. Jackson, D. Hudig, S. L. Woodard, J. C. Powers. 1997. Synthesis and kinetic studies of diphenyl 1-(N-peptidylamino)alkanephosphonate esters and their biotinylated derivatives as inhibitors of serine proteases and probes for lymphocyte granzymes. Arch. Biochem. Biophys. 344:271.[Medline]
  23. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76.[Medline]
  24. Powers, J. C., T. Tanaka, J. W. Harper, Y. Minematsu, L. Barker, D. Lincoln, K. V. Crumley, J. E. Fraki, N. M. Schechter, G. G. Lazarus, K. Nakajima, K. Nakashino, H. Neurath, R. G. Woodbury. 1985. Mammalian chymotrypsin-like enzymes: comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors. Biochemistry 24:2048.[Medline]
  25. Abuelyaman, A. S., D. Hudig, S. L. Woodard, J. C. Powers. 1994. Fluorescent derivatives of diphenyl 1-(N-peptidylamino)alkanephosphonate esters: synthesis and use in the inhibition and cellular localization of serine proteases. Bioconjug. Chem. 5:400.[Medline]
  26. Winkler, U., S. A. Fraser, D. Hudig. 1997. Perforin enhancing protein (PEPr), a low molecular weight protein of cytotoxic lymphocyte granules, enhances perforin lysis. Biochem. Biophys. Res. Commun. 236:34.[Medline]
  27. MacDermott, R. P., R. E. Schmidt, J. P. Caulfield, A. Hein, G. T. Bartley, J. Ritz, S. F. Schlossman, K. F. Austen, R. L. Stevens. 1985. Proteoglycans in cell-mediated cytotoxicity: identification, localization, and exocytosis of a chondroitin sulfate proteoglycan from human cloned natural killer cells during target cell lysis. J. Exp. Med. 162:1771.[Abstract/Free Full Text]
  28. Masson, D., P. J. Peters, H. J. Geuze, J. Borst, J. Tschopp. 1990. Interaction of chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent on pH. Biochemistry 29:11229.[Medline]
  29. Green, G. D. J., E. Shaw. 1979. Thiobenzyl benzyloxycarbonyl-L-lysinate, substrate for the sensitive colorimetric assay for trypsin-like enzymes. Anal. Biochem. 93:223.[Medline]
  30. Schechter, I., A. Berger. 1967. On the size of the active site in proteases. I: Papain. Biochem. Biophys. Res. Commun. 27:157.[Medline]
  31. Winkler, U. 1995. The role of granzymes in perforin-dependent cytotoxicity. Doctoral dissertation, University of Nevada, Reno, NV.
  32. Harper, J. W., K. Hemmi, J. C. Powers. 1985. Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor. Biochemistry 24:1831.[Medline]
  33. Ewoldt, G. R., D. Hudig, P. K. Darcy, J. L. Harris, C. S. Craik, M. J. Smyth. 1997. cDNA cloning of a new granzyme, Granzyme J. Immunogenetics 45:452.[Medline]
  34. Yoshida, N., M. T. Everitt, H. Neurath, R. G. Woodbury, J. C. Powers. 1980. Substrate specificity of two chymotrypsin-like proteases from rat mast cells: studies with peptide 4-nitroanilides and comparison with cathepsin G. Biochemistry 19:5799.[Medline]
  35. Zunino, S. J., N. J. Allison, C. Kam, J. C. Powers, D. Hudig. 1988. Localization, implications for function, and gene expression of chymotrypsin-like proteinases of cytotoxic RNK-16 lymphocytes. Biochim. Biophys. Acta 967:331.[Medline]
  36. Haddad, P., D. Jenne, J. Tschopp, M. V. Clement, D. Mathieu-Mahul, M. Sasportes. 1991. Structure and evolutionary origin of the human granzyme H gene. Int. Immunol. 3:57.[Abstract/Free Full Text]
  37. Tschopp, J., D. Masson, S. Schafer, M. Peitsch, K. T. Preissner. 1988. The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities. Biochemistry 27:4103.[Medline]
  38. Jadot, M., C. Colmant, S. Wattiaux-De Coninck, R. Wattiaux. 1984. Intralysosomal hydrolysis of glycyl-L-phenylalanine 2-naphthylamide. Biochem. J. 219:965.[Medline]
  39. Matsuda, K., S. Ishiura, H. Koizumi, H. Kawasaki, K. Arahata, H. Sugita. 1991. Apolipoprotein B is a major perforin inhibitor protein in human serum. Mol. Immunol. 28:1211.[Medline]
  40. Dupuis, M., E. Schaerer, K. Krause, J. Tschopp. 1993. The calcium-binding protein calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes. J. Exp. Med. 177:1.[Abstract/Free Full Text]



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