Processing of Procarboxypeptidase A and Other Zymogens in

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOL~CICAL CHEMISTRY Vol. 269, No. 41, Issue of October 14, pp. 25897-25904, 1994

Printed in U.S.A.

Processing of Procarboxypeptidase A and Other Zymogens in Murine Mast Cells*

(Received for publication, July 20, 1994)

Michael M. DikovS§, Eric B. SpringmanOfllI, Suresh Yeolan, and William E. SerafinSn** From the $Department of Medicine, Allergy Division, and the Wepartment of Pharmacology, Clinical Pharmacology Division, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232-0111

By cDNA sequence analyses the proteases found within the secretory granules of immunehnflammatory cells appear to be translated initially as zymogens, but by amino-terminal sequencing they are stored within the granules in an active form. We now show that murine mast cell carboxypeptidase A (MC-CPA) is produced in a zymogen form (MC-pro-CPA) that is present at ”0.5% of the level of MC-CPA. MC-pro-CPA is an inactive precursor of MC-CPA and is located within the secretory granules of the mast cells. We have identified one mast cell line, KiSV-MC9, that produces MC-pro-CPA yet cannot process it to the active form despite the fact that these cells can process prochymase and protryptase to their active forms, indicating that a separate mechanism exists for activation of the serine proteases. We show that dipeptidylpeptidase-I is involved in the processing of murine mast cell prochymase and procathep- sin G, but does not process MC-pro-CPA or protryptase. Thus, mast cell carboxypeptidase, tryptase, and chymase zymogens are each processed to their active forms by different mechanisms.

The secretory granules of various immunehnflammatory cells (1-7) contain proteases which are of substantial importance by virtue of their apparent biologic effects and the fact that they constitute a sizable fraction of the total cellular protein of these cells. For example, the secretory proteases constitute up to 50% of the total cellular protein in mast cells (8). Secretory proteases in mast cells include tryptases (g-ll), chymases (9,11,12), and two exopeptidases, mast cell carboxypeptidase A(MC-CPA)’ (13-15) and aminopeptidase (16). Mast cell tryptase, so named because of its trypsin-like activity, has been shown to be a potentiator of histamine-induced bronchocon-

Grants AI31273 and GM15431, a starter grant from the Burroughs * This work was supported in part by National Institutes of Health

Wellcome Fund, and by the Immunology Development Fund, Vanderbilt University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

9 The first two authors contributed equally to this work. 1 1 Supported by a National Institutes of Health Training Grant GM-

07569 and is currently a postdoctoral fellow for the American Lung Association.

**To whom correspondence should be addressed: 847 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0111. Tel.: 615-343-6339; Fax: 615-322-7194.

The abbreviations used are: MC-CPA, mast cell carboxypeptidase A;

transferase; DPPI, dipeptidyl peptidase-I; FLCPA, full-length car- BMMC, bone marrow-derived mast cells; CAT, chloramphenicol acetyl-

boxypeptidase A , Gly-Phe-CHN,, Gly-Phe-diazomethyl ketone; GST, glutathione S-transferase; KiSV-MC, Kirsten sarcoma virus-immortal- ized mast cells; MC-pro-CPA, mast cell procarboxypeptidase A , PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; Fmoc, N-(9-fluorenyl)methoxycarbonyl.

striction (17) and is a fibroblast mitogen (18). Mast cell chymase, possessing chymotrypsin-like activity, is a potent mucous secretagogue when applied to bovine lung epithelial cultures (19). Thus, these mast cell enzymes may be important in the pathogenesis of asthma. In addition to their inflammatory/ immune functions, mast cell proteases may serve as important regulators of vascular tone. MC-CPA has been shown to be a potent inactivator of endothelin-1 by removal of the carboxyl- terminal Trp, with a K,,, for endothelin-1 lower than that reported for any other protease (201, whereas a human mast cell chymase has been found to act as a highly selective angiotensin-converting enzyme producing angiotensin I1 directly from angiotensinogen (21, 22).

An unusual feature of the secretory proteases of immune/ inflammatory cells is that they are stored in fully active form in the secretory granules, rather than as zymogens that are activated after release from the cell. Nevertheless, based on their known cDNA sequences, these immune/inflammatory secretory proteases apparently are initially translated as zymogens. These predicted zymogens are processed a t highly unusual sites compared with other protease zymogens. For example, pancreatic digestive enzymes, the blood clotting cascade, and the complement pathway all include proteases initially produced as zymogens that require removal of amino-terminal segments in order to be activated. These traditional zymogens are activated by clipping the propeptide at a basic residue (typically an Arg) or more rarely at an aromatic residue. In the immunehnflammatory secretory proteases, activation typically occurs by clipping the propeptide at an acidic residue (gran- zymes, chymases, cathepsin G, elastase, carboxypeptidases) or a Gly (mast cell tryptases). A comparison of the processing sites for representative immunehnflammatory proteases is shown in Fig. 1.

Little work has been performed on the activation mechanisms of mast cell proteases. Urata et a l . (49) expressed human chymase in COS-1 cells in three forms: full-length, lacking the putative activation dipeptide, and lacking the leader sequence and the putative activation dipeptide. None of the three forms possessed enzymatic activity. Amino-terminal sequencing of the expressed full-length chymase showed that the leader sequence was appropriately removed by the COS-1 cells, but the putative activation peptide was not. When the recombinant prochymase was incubated with a B-cell lymphoma homogenate, the dipeptide was removed and the chymase became pro- teolytically active. The processing activity present in the B cell lymphoma homogenate was iodoacetimide-sensitive. Thus, this activity likely was due to a thiol protease, but it was not further characterized. McGuire et al. (50, 51) identified an activity termed dipeptidylpeptidase-I (DPPI) present in various granu- lated immune/inflammatory cells. They have proposed that the serine endopeptidases found in lymphocytes, monocytes, and mast cells are activated by proteolytic cleavage of their zymogens by DPPI and have localized the DPPI to the secretory

25897

25898 Mast Cell Procarboxypeptidase A

CARBOXYPEPTIDASE Mouse MC Carboxypeptidase A (-94)4 I E K Q F D V K O E b I A G R . ( 3 0 8 ) Human MC Carboxypeptidase A (-94) I E K Q F D V K E D b I P G R ~r (308) Rat MC Carboxypeptidase A (?) 4 E b I A G R b (308)

CIlYMASElike Mouse MC Protease-l.-2,-4,-L Mouse MC Proteare~S G E b I I G G b (226) Rat MC Protease-I1 E E b I I G G L (227) Human MC Chyrnase Human PMNL Cathepsm G

G E b I I G G * (225)

Human PMNL Elaslase G E b I I G G b (235)

Rat NK Cell Protease-I S E b 1 V G G c (239) G E b I I G G b (228)

Mouse CTL Granryme-C.-D,-E,-F,-G E E b I I G G L (226-232) Mouse CTL Granzynre-B G E b I I G G L (227) Human CTL Granryme-H Human CTL Granzyme-B G E b I I G G c (227)

E E b I I G G b (226)

TRYITASE Mouse MC Pratease~6 A P R P A N Q R V G b I V G G r ( 2 4 5 ) Mouse MC Protease7 Human MC Tryplase a

A P G P A M T R E G ~ I V G G G ( Z ~ ~ )

Human MC lryplase D A P A P V Q A L Q Q A G b l V G G r ( 2 4 6 ) A P A P G Q A L Q R V G b I V G G r ( 2 4 5 )

Dog MC Tryplase Dog Mastocytoma Protease

(-30?) 4 A P G Q A L Q R V G b I V 0 G L (245) (-19?) 4 L L G T L S P K V G b I V G G c (250)

PROPEPTmE b MATURE ENZYME

E E b I I G G b (222-226)

OTHERS Mouse CTL Granzyme-A Human CTL Granzyme-A Rat NK Cell Tryp-2 Rat NK Cell Met-l

E R b I I G G L (232) E K b I I G 0 b (234)

I Y M S S E S F H T E b I I G G r ( 2 3 9 ) N R F E A Q b l I G G h ( 2 3 8 )

FIG. 1. Amino-terminal amino acid sequences for the putative

The point of cleavage between each propeptide and mature amino ter- zymogen forms of various immune/inflammatory cell proteases.

minus is indicated by J . The full extent of each propeptide or mature enzyme is noted in parentheses. The references for the included se-

MC-CPA) (231, human MC-CPA (24), rat MC-CPA (25), mouse MC pro- quences are as follows: mouse mast cell carboxypeptidase A (mouse

teases-1 (261, -2 (27) -4 (28) -L (27), and -5 (29), rat MC protease-I1 (30), human MC chymase (31), human polymorphonuclear leukocyte (PMNL) cathepsin G (6), human polymorphonuclear leukocyte elastase (7), rat natural killer ( N K ) cell protease-1 (32), mouse cytotoxic T- lymphocyte (CTL) granzyme-C (33), -D (34), -E (34), -F (34), -G (35), and -B (36), human CTL granzyme-H (37) and -B (38), mouse MC protease-6 (39) and -7 (40), human MC tryptase-a (41) and -p (42,431, dog tryptase and mastocytoma protease (44), mouse CTL granzyme A (451, human CTL granzymeA(38), rat NK cell tryp-2 (46), and rat NKcell Met-1 (47). Not all amino termini for the mature forms have been determined experimentally; some are by inference from the cDNA sequences. The

van Heijne (48). proposed leader sequence cleavage sites were determined by the rules of

granules of these cells. DPPI is a thiol exopeptidase that re- moves two amino-terminal amino acids at a time from protein and peptide substrates (52, 53). I t shows little substrate specificity, except that it will not cleave carboxyl to a Pro (i.e. Pro in the P, position according to the nomenclature of Schechter and Berger (54)) or if an Arg is located in the P, position (50). Using a covalent inhibitor of DPPI, Gly-Phe-diazomethyl ketone (Gly- Phe-CHN,), McGuire et al. (51) showed that U937 monocytes cultured in the presence of the DPPI inhibitor contained less elastase and cathepsin G activities compared with untreated cells. They examined U937 monocytes for evidence of proca- thepsin G and found increased amounts when the cells were cultured in the presence of the DPPI inhibitor. Using the same DPPI inhibitor, these authors showed diminished tryptic activity in CTL and p815 mast cells (51). These data, and the fact that Urata et al. (49) could activate their recombinant human chymase with a thiol protease, strongly suggest that DPPI is responsible for processing of these serine proteases to their zymogens. The effects of DPPI inhibition on carboxypeptidase and chymase activation were not examined.

We found it surprising that inhibition of DPPI activity would affect activation of mast cell tryptase, since tryptases have activation peptides of 10 or more amino acids (39, 41, 551, rather than dipeptides (Fig. 1). At least one group has reported an endopeptidase activity for DPPI (561, but others, including McGuire et al. (501, have disagreed. If DPPI does process mast cell tryptases by an endopeptidase activity, it might also serve to activate mast cell procarboxypeptidase to its active form. To address whether a single activation mechanism exists for all mast cell secretory granule proteases or different mechanisms

exist depending upon the structure of the propeptide, we have developed methods for identifying mast cell procarboxypeptidase A (MC-pro-CPA) and have characterized its processing to the active form. In order to characterize the effects of DPPI inhibition on the processing of MC-pro-CPA, prochymase, and protryptase to their active forms, we have developed an improved method for the synthesis of the DPPI inhibitor, Gly-Phe-CHN,.

MATERIALS AND METHODS Cell Culture-KiSV-MC14 and KiSV-MC9 were cultured as described

(57). For evaluation of possible constitutive secretion of MC-CPA, KiSV- MC14 and KiSV-MC9 were centrifuged, resuspended in fresh culture medium, and cultured at 5 x lo6 celldml for 0-8 h. Samples were removed every 2 h and centrifuged; the supernatants and cell pellets were evaluated for MC-CPA activity and immunoreactivity (see below). Bone marrow-derived mast cells (BMMC) were raised from murine bone marrow by culture in 50% enriched medium, 50% WEHI-3B-conditioned medium (58) and used during culture weeks 4-6. RBL-1 (CRL 1378), murine NIH-3T3 fibroblasts (CRL 1658), and COS-1 monkey kidney cells (CRL 1650) were obtained from the American Type Culture Collection (Bethesda, MD) and cultured according to their instructions. U937 promonocytes were obtained from Dr. Daniel Tenen (Beth Israel Hospital, Boston) and grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine.

Production ofAntisera to MC-CPAand the MC-CPA Propeptide-MC- CPA was purified from KiSV-MC14 using a potato inhibitor (Sigma) affinity column (13,23).Approximately 20 pg of 98% pure MC-CPAwere obtained per 1 x lo6 KiSV-MC14 as assessed by Bio-Rad protein assay (Bio-Rad) and SDS-PAGWCoomassie Blue staining (23). Antisera to the purified MC-CPA were prepared using New Zealand White rabbits. The presence of anti-MC-CPA antibodies was confirmed by analysis of immunoblots of purified MC-CPA; the working dilution was determined to be 1:20,000 by this method. The specificity of the MC-CPA antiserum was determined by immunoblot analyses of KiSV-MC14 and U937 monocyte sonicates that were resolved by SDS-PAGE. Where indicated purified IgG was obtained from the antiserum using a protein G-agarose affinity chromatography kit (Pierce Chemical Co.).

A fusion protein consisting of glutathione S-transferase (GST) and the MC-CPA propeptide was prepared. A cDNA encoding all 94 amino acids of the murine MC-CPA propeptide was synthesized by the polymerase chain reaction (PCR) using a full-length murine MC-CPA cDNA (23) as the template. Artificial BamHI and EcoRI sites were added to the 5' and 3' ends of the cDNA, respectively, by incorporation of the appropriate sequences within the PCR primers. After restriction enzyme digestion, the cDNA was ligated into the prokaryotic expression plasmid pGEX4XT-1 (59) (Pharmacia Biotech Inc.), which contains a thrombin cleavage site immediately carboxyl to the GST. The plasmid was transfected into Escherichia coli, raised in large quantity, and sequenced across the entire cDNA insert by the method of Sanger et al. (60) as modified for double-stranded templates (61). After induction by the addition of isopropyl-1-thio-p-D-galactopyranoside, the fusion protein was purified from E. coli lysates using a glutathione-agarose (Sigma) affinity column (59). Where indicated, site-specific proteolysis by thrombin (Sigma) was used to cleave the GST portion of the fusion protein from the MC-CPA propeptide (59). The fusion protein (0.2 mg/ ml) and thrombin (0.3 pg/ml) were incubated for 16 h at room temperature in 150 mM NaC1, 50 mM Tris, pH 7.6. Similar methods were employed to produce GST fusion proteins for the active form of murine MC-CPA(containing amino acids 1-308 (using the sequence numbering reported by Reynolds et al. (23)) and for MC-pro-CPA(containing amino acids -94 to 308). Anti-propeptide antisera were prepared in New Zealand White rabbits. The working dilution of the pooled antisera was 1:20,000 when used for immunoblots.

SDS-PAGE and Zmmunoblotting-Pelleted cells or protein samples were suspended in SDS-PAGE sample buffer containing 50 mM dithio- threitol and boiled for 5 min. SDS-PAGE was performed in a discon- tinuous buffer system using 10% resolving gel (23). Prestained protein molecular weight standards (Bio-Rad) were used to calculate apparent molecular weights. Proteins were transferred electrophoretically to polyvinylidene difluoride membranes (PVDF) (DuPont NEN). Mem- branes were blocked with 5% non-fat dry milk, 0.1% Tween 20 in PBS for 45 min, incubated with a 1:20,000 dilution of anti-CPA or anti- propeptide antiserum for 45 min in PBS with 0.3% Tween 20, washed in the same buffer, and finally incubated with a 1:20,000 dilution of peroxidase labeled anti-rabbit IgG antibodies (Boehringer Mannheim) in

Mast Cell Procarboxypeptidase A 25899

the same buffer. Renaissance chemiluminescence reagents (DuPont NEN) and autoradiography film were used according to the manufac- turer's directions to visualize the bound peroxidase conjugate.

Immunofluorescent Staining and Confocal Microscopy-Cells were applied to slides by cytocentrifugation (Cytospin-2, Shandon, Pitts- burgh, PA), air-dried, and fured in Carnoy's solution as described (62). The fixed cells were blocked with 3% BSA, 0.3% Triton X-100 in PBS, probed with 20 pg/ml anti-CPA IgG or anti-propeptide IgG, or nonspecific rabbit IgG (Sigma); stained with Cy-3-labeled anti-rabbit IgG antibodies (Sigma) in blocking solution, and examined with a Zeiss Axiovert 135 confocal fluorescence microscope.

Protease Assays-Carboxypeptidase activity was determined by cleavage of 0.5 mM hippuryl-phenylalanine as assessed by reversed phase-high performance liquid chromatography as described (15) except that multiple time points were obtained for each assay sample and elution was monitored a t 230 nm. The area under the hippuric acid product peak was calculated for each timed sample and compared with a standard curve in order to determine the rate of production of hippuric acid. One unit of carboxypeptidase activity was defined as that amount which cleaved 1 p~ substratdmin.

DPPI activity was determined using Gly-Phe-P-naphthylaminde (Sigma) (51). Conversion to units was determined by comparing the rate of increase of fluorescence intensity with a standard curve prepared using varied amounts of authentic DPPI (Boehringer Mannheim).

Cathepsin G , elastase, and tryptase activities were assayed using succinyl-Ala-Ala-Pro-Phe-p-nitroanalide (63), succiny1-Ala-Ala-Ala-p- nitroanalide (51), and benzoyl-kg-p-nitroanalide as the substrates (64), respectively, with continuous monitoring ofA,,,. Amolar extinction coefficient of 8800 was used for the free p-nitroanalide product. For cathepsin G (63) and elastase (651, the buffer conditions were as described. For tryptase, the buffer was 0.15 M NaC1,2.5 mM CaCl,, 50 mM Tris, pH 7.6, with 0.7 mM substrate. Chymase activity was assayed with 0.5 m~ benzoyl-Tyr-0-ethyl ester (64) in 1 M NaCl, 50 mM CaCl,, 0.2 M Tris, pH 7.6. A molar extinction coefficient of 964 was used for the cleavage product. One unit of activity was defined as that cleaving 1 p~ substrate/min.

Activation of BMMC and Analysis of the Exocytosed Products- BMMC were activated with 0-0.4 p~ calcium ionophore A23187 at 1 x lo7 celldm1 in Tyrode's buffer without gelatin for 20 min at 37 "C as described previously (15). After activation cells were pelleted by cen- trifugation at 200 x g for 10 min at room temperature. The supernatants were removed and the cells were resuspended in Tyrode's buffer and sonicated. Granule content release was monitored by assay of P-hexosaminidase (66). Cell lysis was monitored by following the release of the cytosol marker lactate dehydrogenase (67). The percent release of each cellular constituent was calculated as the amount of the enzyme in the supernatant divided by the sum of the enzyme in the supernatant and the cell pellet. To measure the release of MC-pro-CPA from the activated BMMC, the supernatants were subjected to SDS- PAGE and immunoblotting as described above. The resultant film was scanned with a 2202 Ultroscan (LKB, Bromma, Sweden) densitometer, and the amount of MC-pro-CPA was expressed as area units (OD x mm) under the densitometry trace.

Expression of Recombinant Full-length MC-CPA and MC-CPA Lack- ing the Propeptide in Eukaryotic Cells-Vectors for expression of full- length MC-CPA (FLCPA) and MC-CPA lacking the first 92 amino acids of the propeptide (92delCPA) in mammalian cells were constructed using pRcCMV (Invitrogen, San Diego, CAI. The FLCPA cDNA insert was constructed using PCR as described above. An artificial Hind111 site was inserted immediately 5' to the natural transcription initiation site AAAAAC (23) by inclusion in the sense-strand primer. An artificial 3' XbaI site was inserted after 47 bases of the 3"untranslated region by inclusion in the antisense-strand primer. The cDNA insert for MC-CPA lacking the first 92 amino acids of the propeptide (i.e. containing the leader sequence followed by amino acids -2 to +308) was made using recombinant PCR (68). We chose to leave the COOH-terminal dipeptide Asp-Glu of the propeptide in the construct because we wanted an un- ambiguous leader sequence cleavage site. Preliminary molecular mod- eling had shown the amino terminus of the mature form of MC-CPA to be uninvolved in the overall tertiary structure; and Cole et al. (25) have shown that rat MC-CPA containing an amino-terminal Glu remains active. Artificial 5' Hind111 and 3' XbaI sites were included as described above. The entire nucleotide sequence of each MC-CPA insert, FLCPA and 92delCPA, was verified. For transfections, plasmids were purified on Qiagen-tip 500 columns (Qiagen, Chatsworth, CAI.

E. B. Springman and W. E. Serafin, unpublished observations.

I N-Metiyl morpholine 1. lsobut I Chloroformate

I Piperidine

FIG. 2. Synthesis of Gly-Phe-CHN2

One day prior to transfection, COS (5 x lo5 celldplate) were cultured in 60-mm dishes in fresh medium. On the day of transfection, the COS cells were washed three times with PBS, then incubated for 6 h at 37 "C, 5% CO, in 2 ml of Dulbecco's modified Eagle's medium containing 60 pl of DOTAP reagent (Boehringer Mannheim) and 10 pg of plasmid DNA. After incubation, the transfection solution was removed, 5 ml of fresh COS medium was added to each dish, and the dishes were returned to the incubator. Transfected COS cells were harvested after 48 h.

One day prior to transfection, U937 in log-phase growth were diluted to a concentration of 2 x lo5 celldml in fresh medium. On the day of transfection, the conditioned medium was harvested and 1 x lo7 U937 were washed once with RPMI 1640, then resuspended in 0.5 ml RPMI 1640 and transferred to an electroporation cuvette (0.4-cm gap, Bio- Rad). Plasmid DNA (20 pg) was added, and the cells were left on ice for 10 min. Electroporation was performed using a Bio-Rad gene pulser in two bursts, the first at 300 VI125 microfarads and the second following immediately at 300 V/960 microfarads. Afterwards, the cells were incubated on ice for 10 min. Electroporated U937 were transferred to 10 ml of prewarmed medium (previously conditioned by the overnight culture) and incubated at 37 "C, 5% CO,. Transfected U937 were harvested after 48 h.

The vector pRcCMV/CAT expressing chloramphenicol acetyltrans- ferase (CAT) was a gift from Invitrogen and transfected in parallel samples as a control for transfection success and eficiency. CAT expression in transfected cells was determined using a CAT-enzyme-linked immunosorbent assay (Boehringer Mannheim). Typical transfections in COS and U937 resulted in expression of 8-10 and 1-2 pg of CAT/pg of total protein, respectively. Although expression of the cytosolic protein CAT was maximal in U937 within 24 h, expression of the proteins FLCPA and 92delCPA were maximal at 48-72 h.

Synthesis of Gly-Phe-CHN2-Previously reported synthetic methods either required enzymatic removal of amino protecting groups (69) or resulted in a poorly soluble product with poor inhibitory a~ t iv i ty .~ An improved scheme of synthesis for Gly-Phe-CHN, is shown in Fig. 2. Fmoc-Gly-Phe was purchased from Bachem Bioscience (King of Prussia, PA). Fmoc-Gly-Phe and N-methylmorpholine (0.45 mmol each) were dissolved in 2 ml of dry tetrahydrofuran and cooled to -20 "C. Isobutyl chloroformate (0.45 mmol) was added, and the solution was stirred for 15 min at -20 "C. Cold tetrahydrofuran (10 ml) was added, and the solution was filtered. The filtrate was added to a cold solution of diazo- methane in ether (15 ml). The reaction was kept a t 0 "C for 1 h and then allowed to warm to room temperature. Solvent was removed at reduced pressure, and the Fmoc-Gly-Phe-CHN, was crystallized from methanol/ water. The yield was 0.36 mmol(81%) with a m.p. of 107-109 "C. This compound was characterized by IR (Perkin-Elmer 16PC), 'H NMR (Brucker AC-300), and mass spectrometry (Kratos Concept I1 HH four

M. M. Dikov, E. B. Springman, S. Yeola, and W. E. Serafin, unpublished observations.


A 1 2 3 4 5 1 2 3 4 5 6 7 8 6

FIG. 3. Identification of the zymogen form of MC-CPA. Immu- noblots ofvarious mast cells were analyzed using anti-MC-CPAantisera ( A ) or anti-propeptide antisera ( B ) , a t a 1:20,000 dilution for both. Lanes: 1, KiSV-MC14; 2, KiSV-MC9; 3, BMMC; 4, RBL; 5, U937 monocytes; 6, GST-MC-CPA zymogen fusion protein; 7, GST-active form CPA fusion protein; 8, GST-propeptide fusion protein. The GST fusion proteins were expressed in E. coli and cleaved from the GST using thrombin prior to the SDS-PAGE. MC-CPA expressed in E. coli consistently exhibited a lower molecular weight compared with that of the mast cells. The positions of the molecular weight markers are shown. Lanes 1-5 contained 1.5 x lo5 cells each. Lanes 6-8 contained 0.5 pg of protein each. Detection was by chemiluminescence.

sector tandem mass spectrometer), the m l z was 469 (MH'). Fmoc-Gly- Phe-CHN, (0.21 mmol) was added to 1 ml of piperidine and stirred at room temperature for 30 min. The reaction was poured on cold water (10 ml). The precipitate was filtered and dried under vacuum. The crude product was dissolved in methanol (5 ml), decolorized on charcoal, and recrystallized from methanol to give pure Gly-Phe-CHN,. The yield was 0.16 mmol (76%) with the following characteristics: m.p. 13&139 "C; m l z 247 (MH'); 'H NMR (CDCI,) 8 7.72-7.8 (m, lH), 7.21-7.35 (m, 7H), 5.22 (s, lH), 4.71-4.74 (m, lH), 3.32 (s, 2H), 2.99-3.13 (m, 2H); IR (CHC1.J 3430, 3350, 2112, 1766, 1674 cm".

Inhibition of DPPI Activity in Cultured U937 Monocytes and KiSV- MC14-U937 or KiSV-MC14 cells were cultured as described above, starting at 1.5 x lo5 cells/ml. Gly-Phe-CHN, (3 PM) or diluent (dimethyl sulfoxide, final concentration in culture 0.1%) was added a t day 0, and cells were cultured for 3 days for U937 or 6 days for KiSV-MC14, corresponding to three doubling times for each cell type (70). Additional inhibitor or diluent was added to KiSV-MC14 cells after 3 days to maintain continuous presence of Gly-Phe-CHN, in the 6-day cultures (51). Each day (U937) or every other day (KiSV-MC14), cells were counted and 1.2 x lo6 cells were withdrawn, pelleted, and frozen. After all samples were collected, cells were sonicated a t a concentration of 2 x IO6 cells/ml and assayed for the various protease activities. For sta- tistical comparisons of the starting activity versus the activity following treatment with Gly-Phe-CHN,, Student's t test for paired values was performed.

RESULTS Identification of the Zymogen Form of Mast Cell Carboxypep-

tidase A-Duplicate samples of mast cell lysates were resolved by SDS-PAGE, transblotted to PVDF membranes, and analyzed with either anti-MC-CPA antiserum or anti-propeptide antiserum (Fig. 3). In KiSV-MC14 two prominent bands were seen corresponding to proteins of molecular mass 36 and 52 kDa (Fig. 3A, lane 1 ) using anti-MC-CPA. A less prominent band at approximately 39 kDa was also seen. The 36-kDa protein corresponded to the known size for MC-CPA (231, whereas the 52-kDa protein was somewhat larger in size than the predicted 48.7-kDa size for MC-pro-CPA. The 52- and 39-kDa proteins were detected with the anti-propeptide antiserum, but the MC-CPA band a t 36 kDa was not (Fig. 3B). When an additional KiSV-MC14 lysate was resolved by SDS-PAGE, transblotted to a PVDF membrane, Coomassie Blue-stained, and the resultant 52-kDa band subjected to NH,-terminal amino acid

TARLE I Activities of various proteases in different mast cell types

Enzyme activities are expressed in milliunits/106 cells, n = 3. KiSV-MC14 KiSV-MC9 BMMC

MC-CPA 50,600 f 1800 0.4 2 0.4 3,000 f 400 Chymase 140 2 3 39 t 5 1.6 f 0.2 Tryptase DPPI

0.92 f 0.16 6.8 f 0.02 0.045 f 0.005 27 f. 3 520 f 1 8.6 t 0.4

sequencing according to the technique of Matsudaira (71), the sequence of the first 3 residues was Ile-Ala-Pro, corresponding to the amino-terminal sequence for the propeptide predicted by the cDNA sequence (23).

Serial dilutions of KiSV-MC14 were subjected to SDS-PAGE, and transblots of the dilutions were examined with anti-MC- CPA(not shown). Laser densitometric scanning of the resultant film allowed comparison of the relative levels of MC-pro-CPA and MC-CPA. We scanned different dilutions for MC-pro-CPA and MC-CPA to allow the response for each to be within the linear range of the chemiluminescent technique. By this analysis, 99.5% of the total was present as MC-CPA and 0.5% of the total was MC-pro-CPA. Free propeptide, i.e. after removal from the active form of MC-CPA, was not detected in the KiSV-MC14 despite the fact that recombinant free propeptide was easily detectable (Fig. 3B, lane 8).

KiSV-MC9 Cells Do Not Process MC-pro-CPA to the Active Form, and MC-pro-CPA Does Not Possess Carboxypeptidase Activity-Other populations of cells were examined for expression of MC-CPA and MC-pro-CPA (Fig. 3). By immunoblot analyses, KiSV-MC9, BMMC, and RBL cells contained significant amounts of the 52-kDa MC-pro-CPA (Fig. 31, but only BMMC and RBL, not KiSV-MC9, contained the 36-kDa MC- CPA (Fig. 3A). U937 monocytes contained no immunoreactive proteins using either antiserum.

We next determined the carboxypeptidase activity present in each of the above cell types. KiSV-MC9 cells possess far less carboxypeptidase activity per cell than KiSV-MC14 (Table I). This amount of activity in the KiSV-MC9 is not discernably different from the amount of carboxypeptidase activity present in U937 monocytes (0.7 0.1 milliunits/lOfi cells), which contain no immunoreactive carboxypeptidase (Fig. 3) and do not possess MC-CPA mRNA (not shown). KiSV-MC9 cells do not lack MC-CPA due to their constitutive secretion, because supernatants from cultured KiSV-MC9 cells do not contain MC- CPA activity or immunoreactivity (not shown). As shown in Table I, KiSV-MC9 cells produce substantial amounts of chymase, tryptase, and DPPI activities, indicating that KiSV-MC9 cells do not have a general defect in producingktoring secretory proteases within their secretory granules.

To compare equal microgram amounts of MC-CPA and MC- pro-CPA for carboxypeptidase activity, serial dilutions of KiSV- MC14 and KiSV-MC9 sonicates were prepared, and samples of the dilutions were examined by SDS-PAGE/immunoblotting (not shown). By doing this, we were able to compare levels of MC-CPA in KiSV-MC14 with levels of MC-pro-CPA in KiSV- MC9 and found the ratio to be -1OO:l. We next compared the carboxypeptidase activity found in MC-pro-CPA (KiSV-MC9 sonicate) to that of an equal amount of immunoreactive MC- CPA (KiSV-MC14 sonicate) and found that MC-pro-CPA had -38,000-fold less activity compared with MC-CPA. This indi- cates that MC-pro-CPA is an inactive zymogen.

MC-pro-CPA Is Located in the Secretory Granules of Mast Cells-We examined cytocentrifuge preparations of KiSV- MC14, KiSV-MC9, BMMC, RBL, and U937 monocytes using immunofluorescent staining and confocal microscopy. Staining was performed separately with the anti-MC-CPA and anti-


I I D

FIG. 4. Immunofluorescent staining of BMMC, KiSV-MC9, and transfected U937 assessed by confocal fluorescent microscopy. A, BMMC (anti-MC-CPA IgG); B, BMMC (anti-propeptide); C, KiSV- MC9 (anti-propeptide); D, U937 monocytes transfected with pRcCMV/ 92delCPA (anti-MC-CPA). No specific staining was seen when the primary antibody was absent, when nonspecific IgG was substituted for the primary antibody, or when nontransfected U937 monocytes were used (not shown).

propeptide I&. Each of the mast cell types showed granular staining with both the anti-MC-CPA and anti-propeptide IgG, whereas the U937 cells did not show any specific staining with either IgG. Examples of staining of BMMC and KiSV-MC9 are shown in Fig. 4 and are representative of the other mast cell types. Since KiSV-MC9 cells appear to correctly target MC-pro- CPA to the secretory granules (Fig. 4), the lack of appropriate processing of MC-pro-CPA to its active form in this cell is not due to inappropriate trafficking of the protein.

To confirm that the MC-pro-CPA is located in the secretory granules, BMMC were activated with varied concentrations of calcium ionophore A23187. Release of MC-pro-CPA was compared with release of the secretory granule marker, p-hexosaminidase, and the cytosolic marker, lactate dehydrogenase (Fig. 5). With increasing activation of the BMMC, increasing amounts of MC-pro-CPA were found in the supernatants in parallel with increasing amounts of P-hexosaminidase. In contrast, lactate dehydrogenase content in the supernatants re- mained independent of the cellular activation, consistent with minimal cell deatMysis.

Expression of Recombinant MC-pro-CPA and MC-CPA in Non-mast Cells-When COS-1 cells were transiently transfected with pRcCMVIFLCPA containing the full-length MC- CPA cDNA, they expressed a 52-kDa protein that was immunoreactive with anti-MC-CPA (Fig. 6B, lane 6). Minimal amounts of lower molecular weight bands were seen. No band was seen at 36 kDa, and no carboxypeptidase activity above background amounts was detected (not shown), indicating that COS-1 cells lack the machinery to process MC-pro-CPA to its active form. When pRcCMV/92delCPA, the 92-amino acid deletion mutant lacking all but the two COOH-terminal amino acids of the MC-CPA propeptide, was transfected into the COS-1 cells, a 36-kDa protein was detected by immunoblotting (Fig. 6B, lane 5). However, no carboxypeptidase activity was present above background amounts for these cells (not shown). From this we surmised that one of three possibilities existed. MC-CPA may require the propeptide to fold properly or proper folding can occur only in the unique environment of immune/

0 0.05 0.1 0.2 0.4

A23187, pM

FIG. 5. Release of MC-pro-CPA from the secretory granules of activated BMMC. BMMC were activated with increasing concentrations of calcium ionophore A23187. Top, supernatants from activated cells were analyzed by SDS-PAGE/immunoblotting for content of MC- pro-CPA (PRO and active form MC-CPA using anti-MC-CPA antiserum and chemiluminescence. Bottom, laser densitometry scan results for the MC-pro-CPA band (0) shown in the film in the upper panel. Release of secretory granule contents was monitored by measuring release of the granule marker P-hexosaminidase (A). Nonspecific (cytolytic) release was monitored by measuring release of the cytosolic marker lactate dehydrogenase (*), revealing minimal cell deatMysis (3.5% for unactivated cells). The MC-CPA seen in the supernatants of the unactivated cells represented <3% of the total cellular MC-CPA, similar to that of the lactate dehydrogenase release, indicating this MC-CPA was present due to the minimum cell deatMysis.

A. UP37 monocytes B. COS CdlS

1 0 6 7 80 -:

1 2 3 4 5 6 7

COS kidney epithelial cells ( B ) transfected with with cDNAs for FIG. 6. Immunoblot of human U937 monocytes (A) and monkey

murine MC-CPA. Cells were transfected with either the full-length murine MC-CPA cDNA (pRcCMV/FLCPA, lanes 3 and 6 ) or with an MC-CPA cDNA having the coding region for the first 92 amino acids of the propeptide deleted (pRcCMV/92delCPA; lanes 2 and 5). Controls consisted of U937 and COS cells transfected with pRcCMV/CAT (lanes 1, 4, and 7); 25 ng of authentic MC-CPA were added to the sample in lane 4. Following SDS-PAGE and transblotting, immunostaining was performed with rabbit anti-MC-CPA IgG and developed using chemiluminescence. For both cell types, transfectants containing either pRcCMV/FLCPA or pRcCMV/92delCPA contained <0.1% of their total protein as the recombinant protein.

inflammatory secretory granules, which the COS-1 cells lack, or MC-CPA containing an Asp-Glu dipeptide on the amino terminus maintained the MC-CPA as an inactive zymogen.

We next separately transfected pRcCMVIFLCPA and pRcCMVI92delCPA into U937 monocytes. Both of the recombinant proteins could be successfully expressed in these cells, but the 92-deletion mutant lacking the propeptide was consistently expressed at a substantially higher level. A representative example out of a total of four transfections of U937 cells is shown in Fig. 6A. With pRcCMVFLCPA, a 52-kDa protein consistent with MC-pro-CPA was seen. A small amount of immunoreactive material was also variably seen a t 36 kDa, possibly represent- ing MC-pro-CPA that was processed to the active form. Only minimal amounts of carboxypeptidase activity above back-


2 : # : : r j 3 0.0 4 . 5 E 3

r 5 E 7

2 0 . 1 5

" a & 0.05

0.00

2 3.0 E

v 1.5

0.20

E 0.15

$ 0.10 d 2 0:05 Ei

0.00 0.0 1.0 2.0 3.0

Days FIG. 7. Inhibition of DPPI in US37 monocytes is associated

with inhibition of processing of cathepsin G and elastase to their active forms. U937 myelomonocytic cells were cultured in the presence (0) or absence (0) of the DPPI inhibitor, Gly-Phe-CHN,. Samples were removed daily and assayed for DPPI ( top) , cathepsin G (middle), and elastase (bottom) activities. Enzyme activities are expressed as unitslmg of protein in the cell sonicates.

ground were detected in either transfectant (not shown). By way of comparison, 25 ng of purified MC-CPA (as shown in Fig. 6, lane 4 ) had 265 milliunits of carboxypeptidase activity, in great excess compared with the carboxypeptidase activity present in a similar amount of immunoreactive material from the transfected U937 cells ( 4 milliunit). By immunofluorescent microscopy, a granular distribution was seen in the U937 cells expressing either the FLCPA (not shown) or the 92delCPA (Fig. 4D). From these transfections we concluded that U937 cells do not efficiently process MC-pro-CPA to its active form, despite the fact that they properly direct it to the secretory granules:

MC-pro-CPA, Protryptase, and Prochymase Are Activated by Different Mechanisms-We measured DPPI activity in KiSV- MC14 cells (Table I) and studied the effects of its inhibition on various mast cell protease activities. We synthesized Gly-Phe- CHN, by using a modification of previously reported protocols (69,72) and verified the activity of 0.5 PM in inhibiting purified DPPI as well as DPPI in sonicates of KiSV-MC14 and U937 cells by >95%. When U937 cells were cultured for up to 3 days (-three doubling times) with 3 p~ Gly-Phe-CHN,, the DPPI activity was inhibited by >95% throughout the culture period (Fig. 7). By culture day 2, cathepsin G and elastase activities were diminished by a maximum of 56 and 25%, respectively, consistent with the findings of McGuire et al. (51). When KiSV- MC14 were cultured for up to 6 days (-three doubling times) with 3 p~ Gly-Phe-CHN, (dosing repeated on day 3), the DPPI activity was inhibited by >90% throughout the culture period (Fig. 8). By culture day 4, cathepsin G and chymase activities were diminished by a maximum of 49 and 39%, respectively. In contrast, mast cell tryptase and carboxypeptidase activities were largely unaffected by the inhibition of DPPI (Fig. 8). Rep- etitions of the experiments with the U937 cells and the KiSV- MC14 were performed, and the summary of all of the experiments, including those in Figs. 8 and 9, are presented in Table 11. These data indicate that MC-pro-CPA and tryptase are processed to their active forms by mechanisms distinct from that for chymases.

1 .oo 0 2 4 6

2 c. " Days \ 0 . 7 5 3

0.25

0 . 0 0 - 0 2 4 . 6

FIG. 8. Inhibition of DPPI in KiSV-MC14 is not associated with inhibition of processing of MC-CPA and tryptase to their active forms. KiSV-MC14 were cultured in the presence (0) or absence (0) of the DPPI inhibitor, Gly-Phe-CHN,. Samples were removed every other day and assayed for DPPI, cathepsin G, chymase, MC-CPA, and tryptase activities as indicated. Enzyme activities are expressed as units/mg protein in the cell sonicates.

DISCUSSION

As a general rule, proteases are initially made and stored as inactive zymogens and activated by removal of their NH,-terminal activation segments when their enzymatic activity is needed (73). In contrast to this general scheme, the proteases found in the secretory granules of immunehnflammatory cells are stored in fully active form, and many, e.g. rat mast cell protease-I (74) and MC-CPA (15, 75), have rather broad substrate cleaving capacities. Prevention of inappropriate proteolysis, including autolysis of the cells producing the immune/ inflammatory proteases, appears to be predominantly achieved by sequestration in the secretory granule environment, rather than by retention of an activation peptide. The best candidates for protease regulatory factors in the secretory granule include the low pH, estimated to be 5.5 (76-791, and heparid chondroitin sulfate proteoglycans, which tightly bind the proteases (13,80-82), limiting protease access to one another and to other cellular proteins (74). Although containment within secretory granules may be the predominant factor delimiting inappropriate activity by these enzymes prior to secretion, this is likely not the only important regulatory factor. Unless newly translated proteases are strictly controlled prior to their trans- port to the secretory granules, they could still cause autolysis.

Because of the high degree of similarity of the propeptides for the chymase-like proteases from various immunehnflammatory cells (Fig. l), we and others (1, 7) speculated that these enzymes might share similar activation mechanisms. Thus far, the most promising candidate for a processing activity is DPPI, identified by McGuire et al. (50) to be located in the secretory granules of a variety of immunehnflammatory cells, including mast cells (51, 83). When these investigators cultured various immune/inflammatory cells in the presence of a covalent inhibitor of DPPI, Gly-Phe-CHN,, they could correlate inhibition of DPPI with diminished activities of several of the serine proteases found within the secretory granules of these cells, despite the fact that Gly-Phe-CHN, itself had no direct effect on serine protease activities (51).

We have extended the findings of McGuire et al. (50) to show that DPPI is located in large amounts in mature mast cells


TABLE I1 Effects of Gly-Phe-CHN, on the protease activities in cultured cells

of the activity k S.D. For U937 cells, the values are for day 2 where maximum inhibition of DPPI, cathepsin G, and elastase activities occurred. The values shown are the percent inhibition of the given enzyme activity by culture of cells with Gly-Phe-CHN,, compared with the starting level

Slightly less inhibition was seen for day 3 of the experiments. For KiSV-MC14, values shown are for day 4 where maximum inhibition of DPPI, cathepsin G, and chymase activities occurred. Similar values were obtained for day 6 of the experiments.

DPPI Cathepsin G Elastase Chymase MC-CPA Tryptase

u937 95 -c 1.3" 56 k 9.5" 31 -c 7.5' Not relevant Not relevant Not relevant

KiSV-MC 91 -c 0.4" 43 k 5.3" Not relevant 29 k 7.4" Ok7 4.3 ? 4.1 n = 3 n = 4

n = 3 n = 4 n = 4 n = 3 n = 5

n = 3

" p < 0.01. 'p < 0.05

(Table I) and that it is responsible for the processing of mast cell chymases to their active forms (Fig. 8, Table 11). Since human mast cells have a cathepsin G-like serine protease in their secretory granules (84), we examined murine KiSV-MC14 for a similar activity and found it to be present (Fig. 8). This murine mast cell cathepsin G-like enzyme also appears to be dependent upon DPPI for processing to the active form (Fig. 8, Table 11).

Although the propeptide for MC-pro-CPA is predicted to be far longer than those for the mast cell chymases, both the murine and human MC-CPA propeptides end with 2 acidic amino acids (Fig. 1) (23, 24). We initially postulated that an endopeptidase favoring cleavage at acidic amino acids processed both the mast cell prochymases and procarboxypepti- dases to their active forms (8). However, DPPI would be an unlikely candidate for such an activity. To address this further, we developed methods providing the initial identification of MC-pro-CPA (Fig. 3), found it to be present at low levels in mature mast cells (0.5% of the level of MC-CPA), and characterized it as an inactive precursor of MC-CPA. We have determined that MC-pro-CPA is processed to its active form by a mechanism different from that of the chymases (Fig. 8, Tables I and II), and does not require DPPI activity. Whatever the processing enzyme, it is highly efficient and possibly is located within the secretory granules, since the small amount of MC- pro-CPA that we do find is located within the secretory granules of the mast cells (Figs. 4 and 5 ) .

Because mast cell tryptases also possess activation segments significantly longer than dipeptides (39,41, 551, but were said to be DPPI-dependent for their activation (511, we also examined mast cell tryptase processing in the setting of DPPI inac- tivation. We found no evidence for DPPI being responsible for the activation of protryptase (Fig. 8, Table 11). Thus, mast cell chymases and tryptases are activated by distinct mechanisms. KiSV-MC9 cells can produce MC-pro-CPA but not process it to the active form (Fig. 3). Since KiSV-MC9 have substantial tryptase activity (Table I), we conclude that MC-pro-CPA and protryptase are activated by separate endopeptidase cleav age mechanisms. Thus, we find that mast cell chymases, tryp tases, and carboxypeptidase are activated by three distinct mechanisms.

Using x-ray crystallography, Guasch et al. (85) showed that pancreatic pro-CPA and pro-CPB have highly similar globular propeptides that serve to obstruct the active site of their re- spective mature enzymes. Pancreatic pro-CPB contains a salt bridge between the propeptide and the substrate-binding site of the mature form that precludes docking of substrates, whereas pro-CPA lacks the salt bridge. This difference is thought to explain the fact that pro-CPB is a completely inactive zymogen, whereas pro-CPA retains significant activity toward small substrates (85). Since MC-pro-CPA lacks any enzymatic activity, we predict that it contains a salt bridge similar to that of pancreatic pro-CPB.

The precise fate of the MC-CPA propeptide, once it has been removed from MC-CPA, remains to be determined. Since MC-

CPA constitutes up to 12% of the total cellular protein of mature mast cells (8 ) and the propeptide is fully one-third the length of the mature enzyme, large amounts of propeptide are produced and seemingly quickly discarded, either by degrada- tion or selective secretion. We find no evidence for any free propeptide in KiSV-MC14 (Fig. 3) despite our ability to detect it at levels below 0.5% the level of the mature enzyme form. Since KiSV-MC14 have a doubling time of -48 h (701, they are producing -2% of their MC-CPA/h. Yet, we cannot see any free propeptide in these cells. Since the removal of the propeptide may take place within the secretory granules, we propose that the granules are able to selectively and quickly degrade certain proteins but retain others. Mast cell secretory granules may have evolved from lysozomes (86). The ability of the mast cell secretory granules to rapidly and selectively degrade certain proteins might be considered further evidence for this.

We have provided a substantial methodologic improvement for the synthesis of the DPPI inhibitor Gly-Phe-CHN,. Since McGuire et al. (51) reported the importance of DPPI in the processing of CTL, neutrophil, and mast cell zymogens, there has been considerably increased interest in utilizing this inhibitor. Yet prior synthetic methods were cumbersome, resulted in a final product that was difficult to solubilize and often resulted in an inactive product. By utilizing an Fmoc protecting group for the amino terminus, we subsequently were able to utilize alkaline hydrolysis for its removal, avoiding acid which hy- drolyses the diazomethyl ketone. Gly-Phe-CHN, was produced in large amounts and high punty. More ready availability of this inhibitor should allow more rapid progress in the study of the role of DPPI in immunehnflammatory cells.

Phe-CHN,. Daphne Mitchell provided the BMMC cultures. Julie Acknowledgments-Sam Saleh performed the synthesis of the Gly-

Zaucha performed the initial construction of the 92delCPA expression vector. We thank Dr. John Murray for use of his HPLC and autoinjector, Dr. Ian Blair for helpful comments concerning the synthesis of Gly-Phe- CHN, and for use of the mass spectrometer, Dr. Mary Alice Harbison for use of her spectrofluorimeter, and Tom Jetton for help with the confocal

Vanderbilt Medical Center core facility. The amino-terminal amino acid microscopy. The confocal microscope was made available via the

sequencing of MC-pro-CPA was performed at the Harvard Microchem- istry Facility. Dr. Dwain Thiele (Southwestern Medical Center, Dallas) generously provided the initial samples of Gly-Phe-CHN, from stocks used in his original studies. Dr. Guy Salvesen (Duke University) kindly reviewed the manuscript.

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