Expression and Purification of Recombinant Human Tryptase in a Baculovirus System

PROTEIN EXPRESSION AND PURIFICATION

7, 67–73 (1996)

Article No. 0010

Expression and Purification of Recombinant Human Tryptase in a Baculovirus System1 Kentaro Sakai,* Scott D. Long,* Denise A. Dove Pettit,† Guy A. Cabral,† and Lawrence B. Schwartz*,†,2 *Division of Rheumatology, Allergy and Immunology, Department of Medicine, and †Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia 23298

Received June 21, 1995, and in revised form August 4, 1995

B2 is a mAb that recognizes a conformational determinant on the active form of native tryptase, but does not recognize native tryptase that spontaneously loses activity in physiologic buffer. Precursor forms of recombinant human (rh) a- and rhb-tryptase have been expressed in a baculovirus system. In each case, multiple electrophoretic forms were detected in both culture media and cell lysates of infected insect cells by Western blots developed with the G3 mAb made against native human tryptase. Although only 4 of 30 amino acids in the leader sequences of a- and b-tryptase differ, rha-tryptase appeared predominantly in the cell lysates, rhb-tryptase predominantly in the culture media. B2 recognized rha-tryptase and rhb-tryptase found in the culture media of infected Sf-9 cells, but not that in cell lysates. Secreted forms of tryptase were purified to homogeneity by B2-immunoaffinity chromatography. From 1 liter of culture fluid 1.5 to 3 mg of rh-tryptase could be purified. Each rh-tryptase precursor was enzymatically inactive with synthetic substrates. Analysis of the N-terminal amino acid sequences of purified rha- and rhb-tryptase precursors (APAPVQA and APAPGQA, respectively) indicated that the initial 18 amino acids of the 30-amino-acid leader sequence had been removed. Differential N-glycosylation was found in both rha-tryptase (one or two carbohydrate groups per molecule) and rhb-tryptase (zero or one carbohydrate group per molecule). Thus, the baculovirus expression system is a useful tool for generation of rha- and rhb-tryptase precursors that exhibit a conformational epitope also present on natural tryptase and that are preferentially secreted into the culture media of infected cells. q 1996 Academic Press, Inc.

resides as an enzymatically active enzyme, its activity suppressed by the acid pH of the secretory granule (3) and the high concentration of histamine (4). The active form of tryptase is a tetramer, which is stabilized by its ionic interaction with heparin proteoglycan (1,5,6). The subunits are held together by noncovalent interactions. When separated from heparin and placed in neutral buffered saline, tryptase spontaneously converts to monomers that are inactive. Each subunit also undergoes changes in tertiary and secondary conformations (7). For example, the mouse mAb called B2 recognizes a conformational epitope on the enzymatically active tetrameric form of mature, mast-cell-derived tryptase, but fails to recognize the inactive monomers of this enzyme that form at room temperature in physiologic buffer in the absence of a stabilizing polysacharide like heparin or dextran sulfate. Whether this epitope is available on tryptase precursors has not been addressed. In humans, two cDNA clones called a and b from a lung mast cell library and three cDNA clones called I, II, and III from a skin mast cell library have been identified, each on chromosome 16 (8–10). Tryptase cDNAs b, I, II, and III are at least 98% identical in predicted amino acid sequence to one another. a-Tryptase is about 93% identical to the b-like cDNAs (11,12). The enzymes are synthesized as precursors, which are presumed to be converted into mature forms by limited proteolysis. Recent studies using Northern blotting and/or reverse transcriptase–polymerase chain reaction showed that the predominant type of tryptase 1

Tryptase, a trypsin-like neutral serine protease, appears to be the most abundant protein in the secretory granules of all types of human mast cells (1,2). There it

Supported by National Institutes of Health Grants AI-20487 and DA-05832. 2 To whom correspondence should be addressed at Division of Rheumatology, Allergy and Immunology, Department of Medicine, Virginia Commonwealth University, P.O. Box 980263, Richmond, VA 23298. Fax: (804) 828-0283. 67

1046-5928/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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mRNA expressed by the human monocytic cell lines named Mono Mac 6 and U-937 were a and b, respectively (13); by the human basophil leukemia cell line named KU812 was b (11,12); and by the human mast cell line named HMC-1 was b (12,13). Human skinand lung-derived mast cells express a- and b-tryptase mRNAs, although b-tryptase typically is in excess (12). Peripheral blood basophils express predominantly atryptase mRNA, although cellular levels are several orders of magnitude lower than in mast cells (12). It is not known whether natural tryptase is a homotetramer or heterotetramer, nor whether a- and b-tryptase exhibit distinct enzymatic activities. The baculovirus expression system is a powerful tool that has been used to express high levels of many different functional recombinant eukaryotic proteins, as reviewed in detail in recent books (14–16). The insect cells used in this system, Spodoptera frugiperda (Sf9), perform posttranslational modifications in a fashion similar to mammalian cells. They fold nascent proteins, form disulfide bonds, glycosylate potential N- and Oglycosylation sites (17), and phosphorylate (18), myristoylate (19), palmitoylate (20), and carboxymethylate (21) recombinant proteins. These activities are often essential for functionality. In the present study precursor forms of recombinant human (rh) a- and rhb-tryptase that exhibit the conformational epitope recognized by the B2 anti-tryptase mAb were successfully expressed in serum-free culture media of infected Sf-9 cells with recombinant baculovirus, but not in cell lysates. Both forms of secreted-only rh-tryptase were recognized by the B2 mAb and were purified to apparent homogeneity in high yield by one-step immunoaffinity chromatography with this antibody. MATERIALS AND METHODS

Materials. Bovine serum albumin (BSA), agarose, 2-[N-mopholino]ethane sulfonic acid (MES), 5-bromo4-chloro-3-indoyl phosphate (BCIP)/nitroblue tetrazolium (NBT)-buffered substrate tablet, 2-mercaptoethanol, alkaline phosphatase-conjugated goat IgG antimouse IgG (Fc specific), tosyl-L-arginine methyl ester (TAME), glycerol, ethylene glycol, penicillin, streptomycin, and amphotericin B (Sigma Chemical Co., St. Louis, MO); N-glycosidase F, O-glycosidase, tosyl-L-glycine-L-proline-L-lysine-p-nitranilide (TGPL), BseAI and EcoRI (Boehringer Mannheim Biochemica, Indianapolis, IN); PCR kits and Ampli Wax PCR Gem 50 (Perkin–Elmer Corp., Foster City, CA); l HindIII DNA molecular weight markers and FX174/HaeIII DNA molecular weight makers (Promega Corp., Madison, WI); Coomassie brilliant blue R-250 (Pharmacia LKB Biotechnology AB, Uppsala, Sweden); serum-free S. frugiperda (Sf-9 cells), Sf-900 II serum-free medium for insect cell culture (SFM), and prestained protein molec-

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ular weight standards (Gibco BRL Life Technologies, Inc.,Grand Island, NY); and pVL1392 and pVL1393 transfer vectors, Easy DNA Kit, and AcMNPV Linear DNA Transfer Module containing linearized AcMNPV baculovirus DNA and cationic liposomes (Invitrogen, San Diego, CA) were obtained as indicated. Anti-tryptase mAb, G3, was prepared and purified as previously described (22). Baculovirus PCR primers (5*-TTTTACTGTTTTCGTAACAGT TTTG-3* and 5*-CAACAACGCACAGAATCTAGC-3*) were synthesized and HPLC purified by the Nucleic Acid Core Facility at Virginia Commonwealth University. Cell culture. Sf-9 cells were maintained and propagated either in spinner flasks as a suspension culture or in standard tissue culture flasks as a monolayer culture at 277C in Sf-900 II SFM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. Construction of recombinant baculovirus. cDNA molecules containing the entire coding region of human mast cell a-tryptase (1142 bp) and b-tryptase (1148 bp) were obtained from cDNA clones derived from a human lung cDNA library by digestion with EcoRI (8,10). These coding regions were subcloned into the baculovirus transfer vectors pVL1392 and pVL1393, respectively, by standard techniques. Cotransfections of 1.5 1 106 Sf-9 cells were performed with 1 mg of linearized wild-type AcMNPV baculovirus DNA and 3 mg of pVL1392/a-tryptase (10,781 bp), pVL1393/b-tryptase (10,787 bp), or pVL1392 alone using cationic liposomes as instructed by the manufacturer. After 6 days of incubation of the cotransfection mixtures at 27oC, viruscontaining supernatants were harvested and titered. Viral clones of recombinant baculovirus were purified by three plaque assay screening rounds. Plaques without occlusions were selected for rha-tryptase and rhbtryptase cotransfections to obtain recombinant baculovirus isolates; plaques with occlusions were selected for the pVL1392 cotransfection to obtain a wild-type baculovirus isolate. Purities of the recombinant baculoviruses were confirmed by performing the polymerase chain reaction (PCR) using baculovirus PCR primers (5*-TTTTACTGTTTTCGTAACAGTTTTG-3* and 5*CAACAACGCACAGAATCTAGC-3*) as recommended by Invitrogen, and by digestion of the PCR products with BseAI to distinguish rha- from rhb-tryptase. BseAI cleaves the rhb-tryptase PCR product (1798 bp) into two fragments of 1333 and 465 bp, while the rha-tryptase PCR product (1792 bp) and the wild-type PCR product (650 bp) are not cleaved. The baculovirus genomic DNA was purified using the Easy DNA Kit (Invitrogen) according to the instructions of the manufacturer. The PCR reaction mixtures contained 11 PCR buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM

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MgCl2 , 200 mM dNTP mix) and 1.25 U of AmpliTaq in a final volume of 50 ml. PCRs were performed for 32 cycles using the ‘‘hot start’’ technique with Ampli Wax Gem 50, each cycle including denaturation (947C, 1 min), annealing (557C, 2 min), and extension (727C, 5 min). Each PCR reaction mixture (5 ml) was subjected to electrophoresis in a 1.5% agarose gel containing 0.4 M Tris–acetate buffer, pH 8.3, containing 1 mM EDTA. The gels were stained with ethidium bromide, photographed, and scanned. Purification of recombinant baculoviruses. Re-combinant baculovirus transfer vectors (a-tryptase/ pVL1392, b-tryptase/pVL1393 and pVL1392) were cotransfected with linearized AcMNPV baculovirus DNA into Sf-9 cells to obtain recombinant baculovirus. Each recombinant baculovirus yielded an occlusion-negative plaque and was plaque-purified over three sequential rounds. Wild-type virus yielded occlusion-positive plaques. Purity was confirmed in each case by PCR analysis using baculovirus primers that flank the insertion site and by digestion of the PCR products with BseAI, which cleaves the rhb-tryptase product, but not that of rha-tryptase or wild-type virus. Expression of rha- and rhb-tryptase. High-titer viral stocks (ú108 PFU/ml) were produced by three passages in Sf-9 cells infected at a low multiplicity of infection (m.o.i.) (0.1 PFU/cell). rha-Tryptase and rhb-tryptase proteins were expressed in Sf-9 cells (2 1 106 cells/ well) grown as adherent cell monolayers in six-well tissue culture plates (Falcon) at a high m.o.i. determined to be optimal (5 for rha-tryptase and 2.5 for rhb-tryptase). Sf-9 cells, noninfected or infected with pVL1392 baculovirus at a m.o.i. of 10, were also prepared as controls. The cultures were maintained in Sf-900 II SFM at 277C for 1 to 6 days. After cultivation, cell supernatants were harvested and separated from any detached cells by centrifugation at 400g for 10 min. The cell monolayers were lysed by addition of SDS– PAGE loading buffer [1% SDS, 5% 2-mercaptoethanol (v/v), 5% glycerol, 2.5 mM Tris, pH 8.3, 19 mM glycine, 0.1% (w/v) bromophenol blue]. Detached cells were combined with the cell lysates, sonicated (eight pulses, 50% pulse cycle, 4 power, microtip attachment, Sonicator cell Disruptor Model W-225R; Heat System-Ultrasonics, Inc., Planview, NY), and boiled for 5 min. Proteins in 500 ml of the culture media were concentrated by precipitation in ice-cold 10% trichloroacetic acid, washed with ice-cold acetone, air dried, and resuspended in SDS–PAGE loading buffer. Expression levels of recombinant proteins were monitored by SDS– PAGE (8-cm-long 12% polyacrylamide SDS gels) in a Novex Xcell II Mini-Cell system (Novex, San Diego, CA). Gels were either stained with Coomassie brilliant blue R-250 or subjected to Western blotting and stained with the G3 mAb (0.5 mg/ml) as described below.

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Purification of rha-tryptase and rhb-tryptase. Sf-9 cells (8 x 106 cells) infected with rha-tryptase baculovirus (m.o.i. Å 5, 4 days culture) were extracted by sonication (eight pulses, 50% pulse cycle, 4 power, microtip attachment) in 10 mM MES, pH 6.5, containing 1 M NaCl at 47C and then centrifuged at top speed for 10 min at 47C to remove cell debris. Sf-9 SFM from Sf9 cells infected with rha-tryptase baculovirus (m.o.i. Å 5, 3 days culture, 350 ml) or rhb-tryptase baculovirus (m.o.i. Å 2.5, 4 days culture, 130 ml) was harvested and centrifuged at 400g for 10 min at 47C to remove floating cells. The cell lysate and cell culture media samples were applied to an immunoaffinity column (20ml bed volume) containing B2–Affigel. After loading, the column was washed with 5 column volumes of 10 mM MES, pH 6.5, containing 1 M NaCl and again with 10 mM MES, pH 6.5, containing 0.1 M NaCl. rh-Tryptase bound to the column was eluted with 10 mM diethanolamine, pH 10.0, containing 0.2 M NaCl and 50% (v/v) ethylene glycol as described previously for natural tryptase (7). The eluted fractions were immediately neutralized with 1 M MES, pH 6.5. Chromatographic profiles were monitored by OD280. Deglycosylation of rh-tryptase. Native tryptase from human lung was purified as described previously by B2–agarose immunoaffinity chromatography (7). Immunoaffinity-purified rha-tryptase, rhb-tryptase, and human lung tryptase (10 mg of each) were deglycosylated as follows. Samples were denatured by heating in a boiling water bath for 10 min, cooled to room temperature and incubated with N-glycosidase F (1 U), a mixture of N-glycosidase F (1 U) and O-glycosidase (1 mU), or buffer alone according to the instructions of the manufacturer (Boehringer Mannheim Biochemica). Deglycosylated and unaltered preparations of tryptase were concentrated as described above and subjected to SDS–PAGE. The gels then were stained with Coomassie brilliant blue R-250 and scanned. Western blotting. Samples were subjected to SDS– PAGE under reducing conditions and transferred electrophoretically to a supported precut nitrocellulose membrane (Novex). The membrane then was soaked in 3% BSA to block nonspecific protein binding and incubated overnight at 47C with 0.5 mg/ml of the G3 mAb. Bound antibodies were detected using goat IgG anti-mouse Fcg conjugated with alkaline phosphatase and BCIP/NBT-buffered substrate tablets for color development (Sigma). Prestained protein molecular weight standards (Gibco) were used as molecular weight markers. Analytical techniques. Protein concentrations were measured with the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL) using BSA as a standard (23). Tryptase enzymatic activity was measured by hydrolysis of either TAME or TGPL as described pre-

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FIG. 1. Expression of rh-tryptase in a baculovirus system. Sf-9 cells infected at an m.o.i. of 5 for rha-tryptase or of 2.5 for rhb-tryptase were placed into monolayer cultures. Cell culture media and cells were harvested daily and analyzed by SDS–PAGE (Coomassie brilliant blue staining) and by Western blotting (G3 anti-tryptase mAb). Stained gels and blots were scanned directly and displayed. Molecular weight standards are shown on the left.

viously except that CaCl2 was omitted from the reaction mixtures (24). The N-terminal amino acid sequences of immunoaffinity-purified rha-tryptase and rhb-tryptase (300 pmol of each) were determined by Commonwealth Biotechnologies Corporation (Richmond, VA) using an Applied Biosystems Model 470 gas phase sequencer with a dedicated Model 120A PTHamino acid analyzer. RESULTS AND DISCUSSION

Expression of Recombinant Tryptase To examine expression levels and to optimize expression conditions for each rha-tryptase and rhb-tryptase, a postinfection time course of Sf-9 cells was performed. Sf-9 cells were infected with rha-tryptase baculovirus at an m.o.i. of 5 or with rhb-tryptase baculovirus at an m.o.i. of 2.5. Replicate portions of extracts and media were subjected to SDS–PAGE. One portion was stained directly with Coomassie blue; the other was subjected to Western blotting with the G3 mAb against native human tryptase as shown in Fig. 1. Although only 4 amino acids in the 30-amino-acid leader sequences were different between a- and b-tryptase cDNAs, respectively (S/N028,V/G023, Q/R03, and A/ V02)(8–10), expression characteristics of the corresponding protein products were different in the baculovirus expression system. rha-Tryptase appeared at 2 days postinfection with apparent size heterogeneity consisting of three predominant bands ranging from 29

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to 33 kDa in the cell lysates and of two predominant bands of 29 and 31 kDa in the conditioned media. The G3-immunoreactive bands reached a maximal intensity at 3 days after infection in the culture media and then levels apparently diminished. In the cell lysates maximal staining also was achieved at Day 3 or 4, but remained strong for the entire 6 days of the experiment. The staining intensity of the higher molecular weight band appeared to diminish by Day 6. G3-reactive material from the rhb-tryptase baculovirus infection was more abundant in the culture medium than in the cell lysates and exhibited two predominant bands with apparent molecular weights of 29 and 31 kDa. Immunoreactive material was first detected 3 days postinfection and reached a plateau by Day 4. Also, both in the culture media and in the cell lysates, Western blot analyses showed lower molecular weight bands beginning to appear by Day 4 for rha-tryptase and by Day 5 for rhb-tryptase (data not shown), suggesting that degradation was occurring. Supporting this conclusion was the observation that addition of the cysteine protease inhibitor E-64 to the culture media reduced the intensity of these bands (data not shown). Immunoreactive bands were absent in both conditioned media and cell lysates of noninfected Sf-9 cells or of Sf-9 cells infected with wild-type baculovirus (m.o.i. Å 10)(data not shown). Although the Western blots were more precise for analyzing the time course and relative amounts of tryptase expressed, increased staining intensity of proteins running at the same electrophoretic

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FIG. 2. Purification of rh-tryptase by B2 immunoaffinity chromatography. (A) Cell culture media obtained from rha-tryptase baculovirus cells on Day 3 (350 ml) or (B) rhb-tryptase baculovirus-infected cells on Day 4 (130 ml), were subjected to chromatography on B2–Affigel as described under Materials and Methods. Fractions of 7 ml were collected. To assess purity, 250 ml of input and effluent samples and 50 ml of each eluate fraction were concentrated by precipitation with trichloracetic acid as described under Materials and Methods, subjected to electrophoresis (8-cm-long 12% polyacrylamide SDS gels) and stained with Coomassie brilliant blue R-250. Molecular weight markers are shown to the left of Coomassie brilliant blue-stained polyacrylamide gels containing input (I), effluent (Ef), and eluate fractions. The optical density measurements at 280 nm of corresponding eluate fractions are shown to the left.

mobilities also could be discerned among the Coomassie brilliant blue-stained gels. Purification of rh-Tryptase To purify rh-tryptase, B2 mAb-immunoaffinity chromatography was performed. Based on the time course experiments described above, extraction of Sf-9 cells infected with rha-tryptase baculovirus (m.o.i. Å 5) and collection of the corresponding culture medium were performed on Day 3. For rhb-tryptase, extraction of Sf9 cells and harvesting of culture medium from infected Sf-9 cells (m.o.i. Å 2.5) were performed on Day 4. No attempt was made to purify intracellular forms of rhbtryptase because so little could be detected as shown in Fig. 1. Culture media and cell lysates were applied to B2–Affigel. Coomassie brilliant blue-stained SDS– polyacrylamide gels of input (I), effluent (Ef), and eluate (elution numbers 1 through 6) fractions are shown in Fig. 2 for the cell media alongside the OD280 measurements of the eluate fractions. rha-Tryptase and rhb-tryptase from culture media, but not rha-tryptase from cell extracts (not shown), bound to the B2–Affigel column and were successfully purified, as shown by the disappearance in effluent fractions of the recombinant bands of protein migrating near 30 kDa, and their ap-

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pearance in eluate fractions 3 and 4. Using serum-free media for Sf-9 cell cultures reduced levels of extraneous protein and thereby facilitated the purifications. Estimates of the portion of total protein due to rha- and rhb-tryptase molecules in the culture media of infected Sf-9 cells were 7% (3 days culture) and 16% (4 days culture), respectively, by densitometry of Coomassie brilliant blue-stained SDS–polyacrylamide gels. The yields of rha-tryptase and of rhb-tryptase were approximately 1.5 and 3 mg/liter of culture media, respectively. The secreted forms of rha-tryptase and rhb-tryptase are conformationally correct with respect to the B2 conformational epitope. This epitope is not available to the B2 mAb on intracellular forms of rha-tryptase, even though the apparent sizes of intracellular and extracellular forms appear to be similar. The presence of the B2 conformational epitope on enzymatically inactive tryptase precursor may reflect correct folding of the precursor, but further studies will be needed to examine this hypothesis. Although enzymatically active, native tryptase is a tetramer, preliminary gel filtration experiments indicate recombinant tryptase precursors are monomers. Native mature tryptase spontaneously inactivates, becomes monomeric, and loses the B2 epi-

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FIG. 3. Glycosylation and N-terminal amino acid sequence of rh-tryptase. (A) B2 immunoaffinity-purified rha-tryptase, rhb-tryptase, and natural lung-derived tryptase were subjected to Western blotting with G3 anti-tryptase mAb before (0) and after (/) deglycosylation with N-glycosidase F. The position of the 30,000 molecular weight marker is shown to the right. (B) N-terminal amino acid sequences determined in the current study for purified rha- and rhb-tryptase preparations, the full-length sequences predicted from the recombinant cDNA molecules utilized, and the previously determined sequence for mature lung-derived tryptase. The presence (/) or absence (0) of detectable enzymatic activity with either TAME or TGPL substrates is shown to the right of the amino acid sequence information. Parentheses indicate predictions rather than direct measurements.

tope when separated from heparin and placed in neutral buffered saline (6,7). However, both rha-tryptase and rhb-tryptase in the culture media of infected Sf-9 cells retained the B2 epitope during culture at 277C in the absence of added heparin, reflecting either their precursor stage or the presence of protective molecules that like heparin have a high negative charge density. Deglycosylation of rh-Tryptase Because Sf-9 cells permit posttranslational glycosylation of recombinant proteins, differential glycosylation was evaluated to explain the apparent size heterogeneity observed in Figs. 1 and 2. Immunoaffinity-purified rha- and rhb-tryptase molecules were treated with N-glycosidase F and then subjected to SDS–PAGE as shown in Fig. 3. The two major bands of rha-tryptase and of rhb-tryptase were reduced to a single band after deglycosylation. Based on Pharmacia low molecular weight protein standards, the two major bands of purified rha-tryptase exhibited molecular weights of 31.0 and 30.3 kDa. After deglycosylation they were reduced to the molecular weight of a minor band seen at 29.4 kDa. rhb-Tryptase after deglycosylation was reduced from two bands at 30.7 and 30.2 kDa to the lower molecular weight band at 30.2 kDa. a-Tryptase cDNA encodes two potential N-linked carbohydrate binding regions, whereas b-tryptase cDNA encodes only one potential N-linked carbohydrate binding region (8,10). This may account for much of the size heterogeneity of recombinant tryptase. Of the two major bands of rha-

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tryptase, the one with a slower electrophoretic mobility may have two carbohydrate groups; the other may have only one carbohydrate group. The two forms of rhbtryptase may differ by having either one or zero carbohydrate groups per molecule. In contrast, the slower, more diffuse bands of native human lung tryptase were reduced to two bands (30.5 and 29.5 kDa) after deglycosylation, the lower molecular weight band being predominant. When using a combination of N-glycosidase F and O-glycanase for deglycosylation of rh-tryptase molecules and lung-derived tryptase, similar results to a single digestion of N-glycosidase F were obtained (data not shown), suggesting that O-linked carbohydrates were not present. N-glycosylation may not be necessary for correct folding of tryptase to occur during processing, because nonglycosylated molecules of both rh-tryptase molecules appear to be recognized by the B2 mAb. The first seven NH2-terminal amino acid residues in immunoaffinity-purified rha-tryptase and rhb-tryptase were determined as shown in Fig. 3. In each case the sequence began at Ala012 of the leader peptide; Met030 –Ala013 had been removed. Neither immunoaffinity-purified rha- nor rhb-tryptase had detectable enzymic activity with synthetic substrates (TGPL and TAME), consistent with the enzyme being in a precursor form. Also, when cell extracts or cell media were examined, no tryptase activity could be detected. To our knowledge, generation of enzymatically active rhtryptase has not been reported, but is now possible to

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study using the precursor tryptase molecules described in the current study. Recent studies suggest that mast cell proteases associated with secretory granules such as chymase, cathepsin G, and carboxypeptidase A are processed in part by dipeptidyl peptidase I (25–27). Less information on the processing of tryptase is available. In rodents dipeptidyl peptidase I involvement in tryptase processing has been suggested in one study (27), but lack of involvement was found in another study (26). Of possible signficance is that the Ala-Pro N-terminus of the recombinant tryptase molecules analyzed in the current study is a stop sequence for dipeptidyl peptidase I.

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REFERENCES 1. Schwartz, L. B., Lewis, R. A., Austen, K. F. (1981) Tryptase from human pulmonary mast cells. Purification and characterization. J. Biol. Chem. 256, 11939–11943. 2. Schwartz, L. B., Irani, A. M. A., Roller, K., Castells, C., and Schechter, N. M. (1987) Quantitation of histamine, tryptase and chymase in dispersed human T and TC mast cells. J. Immunol. 138, 2611–2615. 3. Lagunoff, D., and Rickard, A. (1983) Evidence for control of mast cell granule protease in situ by low pH. Exp. Cell Res. 144, 353– 360. 4. Alter, S. C., and Schwartz, L. B. (1989) Effect of histamine and divalent cations on the activity and stability of tryptase from human mast cells. Biochim. Biophys. Acta 991, 426–430. 5. Alter, S. C., Metcalfe, D. D., Bradford, T. R., and Schwartz, L. B. (1987) Regulation of human mast cell tryptase. Effects of enzyme concentration, ionic strength and the structure and negative charge density of polysaccharides. Biochem. J. 248, 821–827. 6. Schwartz, L. B., and Bradford, T. R. (1986) Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer. J. Biol. Chem. 261, 7372–7379. 7. Schwartz, L. B., Bradford, T. R., Lee, D. C., and Chlebowski, J. F. (1990) Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer: Production of monoclonal antibodies recognizing active tryptase. J. Immunol. 144, 2304–2311. 8. Miller, J. S., Westin, E. H., and Schwartz, L. B. (1989) Cloning and characterization of complementary DNA for human tryptase. J. Clin. Invest. 84, 1188–1195. 9. Vanderslice, P., Ballinger, S. M., Tam, E. K., Goldstein, S. M., Craik, C. S., and Caughey, G. H. (1990) Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family. Proc. Natl. Acad. Sci. USA 87, 3811–3815. 10. Miller, J. S., Moxley, G., and Schwartz, L. B. (1990) Cloning and characterization of a second complementary DNA for human tryptase. J. Clin. Invest. 86, 864–870. 11. Blom, T., and Hellman, L. (1993) Characterization of a tryptase

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