Human α- and β-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. β-Protryptase is sequentially processed in vitro by autocatalysis at R−3 followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R−3Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. β-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q−3) and mutated β-protryptase (R−3Q) as well as wild-type β-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and β-protryptases from human mast cells to their mature enzymatically active products.

β-Tryptase (EC 3.4.21.59), a serine protease, is the major protein component of the secretory granules of human mast cells (1, 2). β-Tryptase is expressed by the monomorphic TPSB2 locus on human chromosome 16p13.3, whereas the adjacent dimorphic TPSAB1 locus encodes either β-tryptase or α-tryptase. The 12 aa propeptide of β2-protryptase can be sequentially processed in vitro to mature β2-tryptase by autoproteolytic cleavage next to R−3 (at pH 6 in the presence of heparin) followed by removal of the remaining two amino acids by cathepsin C (dipeptidylpeptidase I) (3). Processing of the propeptide directly by cathepsin (CTS) C is prevented by Pro−11 in the P1 position, which serves as a stop signal for this dipeptidase. The newly formed mature β-tryptase in the presence of heparin at acidic pH spontaneously aggregates into tetramers that exhibit strong proteolytic activity. Because each subunit of the tetramer faces the inner core of the planar tetramer (4), high m.w. substrates and inhibitors have limited access to these active sites. The autocatalytic–CTSC processing pathway for β-tryptase has been supported by experiments with human mast cell leukemia cell-1 line (HMC-1) cells, a human mast cell leukemia cell line that encodes β1- and β3- but not α-tryptases (5), showing that Gly-Phe-CHN2, an inhibitor of CTSC, attenuates the formation of tryptase activity (3). However, mast cells from mice that are CTSC deficient express mature murine mast cell protease (MMCP)-6, a murine tryptase, albeit at cellular levels ∼75% lower than wild-type cells, indicating the presence of one or more processing pathways for which CTSC is not required (6). This raises the possibility of alternative processing enzymes for β-protryptase in humans.

α-Tryptase is expressed by TPSAB1 in ∼75% of individuals; 25% being α-tryptase deficient, as are HMC-1 cells (79). Unlike β-protryptase, α-protryptase has Q in the −3 position, which makes the propeptide resistant to processing by tryptic enzymes (3). Further, mature recombinant α-tryptase made experimentally exhibits essentially no proteolytic activity and minimal peptidolytic activity because D215 (rather than G present in most serine proteases) places the D side chain into the substrate binding pocket, thereby restricting entry of larger substrates (1013). Whether α-protryptase is processed to mature α-tryptase in vivo and, if so, by what mechanism remain to be determined.

Cathepsins are classically considered lysosomal proteases involved with the degradation of proteins. However, examples of additional cell-specific duties have emerged. By removing dipeptides, CTSC processes progranzymes A, B, and K in CTLs and NK cells (1417), proCTSG (18), proelastase, and proP3 in neutrophils (19), and prochymases in murine mast cells (6). CTSE processes mast cell procarboxypeptidase in murine mast cells (20). However, specific protryptase processing enzymes outside of the autocatalytic–CTSC pathway have not been identified.

The current study uses ion exchange and gel filtration chromatography to purify partially human protryptase processing activities from HMC-1 cells, tandem mass spectroscopy to identify the specific protease candidates, and biochemistry to characterize their processing activities in solution. Using this approach, CTSB and CTSL are shown to be potent and direct processing enzymes not only for human β-protryptase but also for human α-protryptase.

Anti-tryptase mAb G3 for Western blotting was used as described (3, 13). The human mast cell leukemia cell line HMC-1 was provided by Dr. G. Gleich and Dr. J. Butterfield (Mayo Clinic, Rochester, MN) (21). CLIK-148 (CTSL inhibitor), CLIK-060 (CTSS inhibitor), and CA-074 (CTSB inhibitor) (22, 23) were provided by Prof. Nobuhiko Katunuma. Bovine spleen CTSC and human CTSs (CTSL, CTSB, CTSG, CTSZ/P/X, and CTSD) were purchased from Sigma Chemical Co. (St. Louis, MO).

β-Protryptase (500 ng) was incubated in 100 μl 0.01 M Mes, pH 6, containing 0.1 M NaCl, 25 μg/ml heparin, 1 mM EDTA, 5 mM DTT, and 0.1 mg protein/ml of HMC-1 cell extract or with specific proteases at 37°C as specified further in the 7Results, and the incubation mixtures were then assessed for tosyl-l-Gly-Pro-Lys-p-nitroanilide (TGPK) catalytic cleaving activity as described (24). These TGPK assays contained soybean trypsin inhibitor (10 μg/ml) to inhibit serine proteases other than tryptase. In some cases, β-pro′tryptase was generated by preincubating β-protryptase with heparin and 0.5 mg/ml BSA in acetate buffer, pH 6, at room temperature for 30 min as described (3). Terminal amino acid sequencing analyses were performed by Edman degradation on an ABI Procise sequencer by Alphalyse (Palo Alto, CA).

All purification steps were carried out at 4°C. All chromatography steps except the carboxymethyl (CM)-cellulose and sulfopropyl (SP)-sepharose steps were carried out using a Shimadzu HPLC (SCL-10Avp) system (Shimadzu). HMC-1 cells (∼2 × 108 cells) were washed and sonicated on ice while in 50 ml of 0.01 M Mes, pH 6.5, containing 0.1 M NaCl (buffer A). Soluble β-protryptase processing activity was obtained by centrifugation (12,000 × g for 20 min at 4°C) of the disrupted cells, after which the supernatant was applied to a CM-cellulose column (100-ml bed volume), pre-equilibrated with buffer A. The flow-through fractions were collected, desalted (PD-10 chromatography), brought to pH 5.2 with 1 N acetic acid, and then applied to SP-Sepharose (100 ml bed volume) equilibrated with 50 mM sodium acetate buffer, pH 5.2 (buffer B). Most of the CTSC activity was retained, whereas β-protryptase processing activity was also recovered in the effluent. Protein in these latter fractions was precipitated by cold acetone (90%), centrifuged (12,000 × g for 20 min at 4°C), and resuspended in 50 mM Mes buffer, pH 6 (buffer C). This SP effluent was loaded onto a Mono Q 5/5 column equilibrated in buffer C, which was then washed with 10 bed volumes of buffer C containing 0.2 M NaCl to remove any remaining CTSC, after which the bound β-protryptase processing activity was eluted with a linear gradient of 0.2 to 0.7 M NaCl with a flow rate of 1 ml/min. Eluate fractions were collected, concentrated by centricon YM 10 ultrafiltration, and then loaded onto Superpose 12 10/300 gel filtration column (24 ml bed volume). Fractions of 1 ml were collected and assayed for β-protryptase processing activity. Active gel filtration fractions were pooled, reloaded onto a Mono Q 5/5 column equilibrated in buffer C, washed with buffer C, and then eluted with a linear gradient of 0 to 0.8 M NaCl.

Fractions that contained β-protryptase processing activity were collected, concentrated, and subjected to mass spectrometry (W.M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia, Charlottesville, VA). Samples were adjusted to pH 8, reduced with DTT and alkylated with iodoacetamide, digested with trypsin, and acidified with acetic acid. Liquid chromatography–tandem mass spectrometry was then performed by injecting samples onto a Finnigan LTQ-FT mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm (height) × 75 μm (internal diameter) Phenomenex Jupiter 10 μm C18 reversed-phase capillary column. Full scan mass spectra were acquired to determine peptide molecular weights, and then product ion spectra were acquired to determine amino acid sequences in sequential scans. Data were analyzed using the Sequest search algorithm against human IPI.

Human recombinant β2-protryptase and α-protryptase were prepared as described (25). R−3Q β2-protryptase was prepared from human β2-preprotryptase cDNA inserted into the pBluescript II SK vector (Stratagene, La Jolla, CA), which was mutated to convert R−3 to Q−3 (R−3Q) using the site-directed mutagenesis protocol recommended by the manufacturer (QuickChange Site-Directed Mutagenesis Kit) by changing the −3 codon from CGA to CAA. DNA coding for R−3Q βII-tryptase was cloned into the pFastBac 1 vector (Invitrogen) and used to transform DH10 Bac Escherichia coli (13). α-Protryptase, β-protryptase, and R−3Q β-protryptase inserted into a baculovirus expression vector were expressed in and secreted by insect cells and were immunoaffinity purified from the medium by B2-agarose (25). Mature human lung tryptase was purified by B2-agarose immunoaffinity and heparin-agarose chromatography as described (26), and β1/3-protryptases were purified from HMC-1 culture medium by B2-agarose chromatography.

Activities of CTSC (Gly-Phe-β-naphthylamide) (27), CTSL (Z-Phe-Arg-7-amido-4-methylcoumarin [MCA] substrate), and CTSB (Z-Arg-Arg-MCA) (28) were measured as described. For inhibition studies, each enzyme was preincubated with various inhibitors at room temperature for 15 min, and activity was measured. Protein concentrations were measured with the BCA method using BSA as a standard. G3 mAb, which recognizes both pro and mature forms of α- and β-tryptases (25), and mAbs against CTSL and CTSB (Sigma) and against CTSC (R&D Systems) were used for Western blotting and detected with IRDye-conjugated anti-mouse IgG (Odyssey Infrared Imaging System; LiCor Biotechnology, Lincoln, NE).

Tryptase samples were incubated in 1% SDS, 125 mM Tris, pH 6.8, and 10% glycerol in the absence of a reducing agent for 10 min at room temperature and then subjected to SDS-PAGE. In one case, 10% polyacrylamide was copolymerized with a 0.2 mM Boc-Gln-Gly-Arg-MCA (Peptides International, Louisville, KY) as described (29). After electrophoresis, the gel was washed twice in 2.5% Triton X-100 for 20 min to remove SDS and then incubated at 37°C for 30 min in 100 mM Tris pH 7.5 buffer containing 25 μg/ml heparin, 0.005% Brij-35, and 0.15 M NaCl. Hydrolysis of the peptide–MCA substrate by the protease released fluorescent 7-amino-4-methyl-coumarin, which was observed and photographed under a UV transilluminator. In the other case, gelatin zymography was performed in a 10% polyacrylamide gel that had been copolymerized with 0.1% gelatin (30). After electrophoresis, the gel was washed as above and then incubated overnight at 37°C before staining with Coomassie blue. In neither type of zymography was there evidence for CTSB or CTSL activity, presumably because the running, incubation, and detection conditions were not conducive to their stability and activity.

Extracts of HMC-1 cells were subjected to ion exchange fractionation to identify potential β2-protryptase processing enzymes distinct from CTSC (Fig. 1A). CM-Sepharose was used to debulk most of the protein. Approximately 94% of the protein bound under the loading and washing conditions used. But all β2-protryptase processing activity was detected in the effluent. SP-Sepharose chromatography yielded processing activity in the effluent and one peak of processing activity in the salt gradient of the eluate. The eluate peak of processing activity appeared to consist of only CTSC (see later), whereas the effluent peak, though sometimes containing a portion of CTSC, appeared to contain non-CTSC processing activity. Mono Q chromatography of the SP effluent yielded two peaks of activity in the salt gradient eluate, one at ∼200 mM NaCl that consisted of CTSC (see later), and another at ∼450 mM NaCl that did not contain detectable CTSC. This non-CTSC peak yielded one peak of activity after Superose 12 chromatography with an apparent molecular mass of 30 kDa and could be concentrated and further purified by a second Mono Q chromatographic step (Fig. 1B). The Mono Q eluate was then subjected to liquid chromatography–tandem mass spectrometry after trypsin digestion. Informative peptides identified several proteases (Fig. 1C).

FIGURE 1.

Identification and partial purification of processing enzymes for β-protryptase as CTSL and CTSB in HMC-1 cells. A, Overview of the FPLC separation procedure for identifying β2-protryptase processing enzyme(s). Soluble proteins in HMC-1 cell extracts were sequentially subjected to chromatography on CM-Sepharose, SP-Sepharose, Mono Q, and Superpose 12 columns as described in 1Materials and Methods. CTSC activity appeared in the SP-Sepharose eluate; other candidate processing enzymes in the effluent. B, Mono Q anion-exchange chromatography. The Superose 12 peak of β-protryptase processing activity was adjusted to 0.05 M Mes, pH 6, without NaCl; the Mono Q column was loaded and washed with this buffer, and then a linear gradient of 0–0.8 M NaCl was applied. Fractions with β-protryptase processing activity were identified at 0.4–0.5 M NaCl, collected, and analyzed by liquid chromatography–tandem mass spectrometry. C, Proteases identified by mass spectrometry. Informative peptide sequences revealed CTSL, CTSB, CTSZ, and dipeptidyl peptidase III (DPPIII) as candidate processing proteases.

FIGURE 1.

Identification and partial purification of processing enzymes for β-protryptase as CTSL and CTSB in HMC-1 cells. A, Overview of the FPLC separation procedure for identifying β2-protryptase processing enzyme(s). Soluble proteins in HMC-1 cell extracts were sequentially subjected to chromatography on CM-Sepharose, SP-Sepharose, Mono Q, and Superpose 12 columns as described in 1Materials and Methods. CTSC activity appeared in the SP-Sepharose eluate; other candidate processing enzymes in the effluent. B, Mono Q anion-exchange chromatography. The Superose 12 peak of β-protryptase processing activity was adjusted to 0.05 M Mes, pH 6, without NaCl; the Mono Q column was loaded and washed with this buffer, and then a linear gradient of 0–0.8 M NaCl was applied. Fractions with β-protryptase processing activity were identified at 0.4–0.5 M NaCl, collected, and analyzed by liquid chromatography–tandem mass spectrometry. C, Proteases identified by mass spectrometry. Informative peptide sequences revealed CTSL, CTSB, CTSZ, and dipeptidyl peptidase III (DPPIII) as candidate processing proteases.

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Commercial preparations of human CTSL, CTSB, and CTSC could each generate active β2-tryptase from β2-protryptase at pH 6 in the presence of heparin (Fig. 2A). However, other CTSs including CTSD, CTSZ/P/X, CTSS, and CTSG, and dipeptidylpeptidases II and III did not. CTSC removes the residual VG dipeptide from the N terminus of β-pro′tryptase following the heparin-dependent autocatalytic cleavage at R−3 to remove 10 of the 12 aa of the propeptide; mature β-tryptase spontaneously forms the active tetramer at acidic pH in the presence of heparin, which once formed is stabilized at both neutral and acidic pH by heparin (3, 24, 31). Because β2-protryptase is monoglycosylated and β1- and β3- protryptases are diglycosylated, CTSL and CTSB were tested with protryptase(s) that had been purified from the medium of cultured HMC-1 cells, which only carry the genes that encode β1- and β3-tryptases. As shown in Fig. 2B, these CTSs also activated these diglycosylated β-protryptases.

FIGURE 2.

Activation of β-protryptases by CTSL, CTSB, and CTSC. A, β2-Protrypase is selectively activated by CTSB, CTSL, and CTSC. Various proteases were incubated with β2-protryptase (5 μg/ml) in the presence of heparin (50 μg/ml) in 50 mM sodium acetate buffer, pH 5.5, containing 150 mM NaCl, 1 mM EDTA, 5 mM l-cysteine, and 5% glycerol for 30 min at 37°C. B, β1/3-Protryptase(s) were incubated with CTSL and CTSB. Protryptase in the medium from HMC-1 cultures was purified by B2-agarose immunoaffinity chromatography and incubated with CTSB, CTSL, or buffer alone along with heparin and assessed for tryptase activity as in A. C and D, Activation of β2-protryptase by CTSL (C) and CTSB (D) in the presence of specific protease inhibitors. β2-Protryptase was activated with CTSL or CTSB that had been preincubated alone or with the protease inhibitors as shown; micromolar concentrations of inhibitors are shown in parentheses. Activation of β2-protryptase was measured with TGPK. In the absence of inhibitor, β2-protryptase processing activity was set at 100%. The results shown are the average of three independent experiments. E, Inhibition of β2-protryptase processing activity obtained from HMC-1 cells. The CM-Sepharose effluent, which contained all β2-protryptase processing activity, was examined for processing activity in the presence of inhibitors of CTSL, CTSB, and CTSC, each at 10 μM, in three independent experiments. II, dipeptidylpeptidase II; III, dipeptidyl peptidase III; B, CTSB; C, CTSC; D, CTSD; G, CTSG; L, CTSL; S, CTSS; Z, CTSZ/P/X.

FIGURE 2.

Activation of β-protryptases by CTSL, CTSB, and CTSC. A, β2-Protrypase is selectively activated by CTSB, CTSL, and CTSC. Various proteases were incubated with β2-protryptase (5 μg/ml) in the presence of heparin (50 μg/ml) in 50 mM sodium acetate buffer, pH 5.5, containing 150 mM NaCl, 1 mM EDTA, 5 mM l-cysteine, and 5% glycerol for 30 min at 37°C. B, β1/3-Protryptase(s) were incubated with CTSL and CTSB. Protryptase in the medium from HMC-1 cultures was purified by B2-agarose immunoaffinity chromatography and incubated with CTSB, CTSL, or buffer alone along with heparin and assessed for tryptase activity as in A. C and D, Activation of β2-protryptase by CTSL (C) and CTSB (D) in the presence of specific protease inhibitors. β2-Protryptase was activated with CTSL or CTSB that had been preincubated alone or with the protease inhibitors as shown; micromolar concentrations of inhibitors are shown in parentheses. Activation of β2-protryptase was measured with TGPK. In the absence of inhibitor, β2-protryptase processing activity was set at 100%. The results shown are the average of three independent experiments. E, Inhibition of β2-protryptase processing activity obtained from HMC-1 cells. The CM-Sepharose effluent, which contained all β2-protryptase processing activity, was examined for processing activity in the presence of inhibitors of CTSL, CTSB, and CTSC, each at 10 μM, in three independent experiments. II, dipeptidylpeptidase II; III, dipeptidyl peptidase III; B, CTSB; C, CTSC; D, CTSD; G, CTSG; L, CTSL; S, CTSS; Z, CTSZ/P/X.

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To validate that it is the CTSs rather than impurities that process β-protryptase, the effects of selective CTS inhibitors on processing were examined. As shown in Fig. 2C and 2D, β2-protryptase processing by CTSL was inhibited by the general cysteine protease inhibitor E-64 and the specific CTSL inhibitor CLIK-148, but not by the CTSB inhibitor CA-074 and CTSC inhibitor Gly-Phe-CHN2, and processing by CTSB was inhibited by E64 and CA-074 but not by CLIK-148 and Gly-Phe-CHN2. Thus, inhibitor data confirm CTSB and CTSL each process β-protryptase to the mature enzyme.

What portions of β-protryptase processing activity in HMC-1 cells are accounted for by CTSB, CTSL, and CTSC was considered. The CM-Sepharose effluent, which contained all detectable β2-protryptase processing activity, was incubated with various combinations of specific CTS inhibitors and then tested for β2-processing activity. As shown in Fig. 2E, inhibition of CTSL, CTSB, and CTSC reduced β2-processing activity by 43, 34, and 30%, respectively. A combination of CTSL and CTSB inhibitors reduced β2-processing activity by 80%, and a combination of all three inhibitors reduced this activity by 98%. Thus, by this in vitro assessment of β2-processing activity, these three CTSs account for nearly all processing activity.

The generation of tryptase activity from β-protryptase by CTSL and CTSB was also dependent on heparin (Fig. 3A, 3B, respectively). In its absence, no tryptase activity could be detected, and neither chondroitin sulfate A nor B with their lower negative charge densities could effectively replace heparin. The heparin dose-response was similar for CTSL and CTSB, 25–300 μg/ml heparin was optimal with 3.5–4.5 μg/ml β-protryptase and 3–4 μg/ml of either CTS.

FIGURE 3.

Glycosaminoglycan dose-response effects on β2-protryptase activation by CTSB (A) and CTSL (B). Each CTS was incubated with β2-protryptase and various concentrations of heparin, chondroitin sulfate A, and chondroitin sulfate B in sodium acetate buffer as for Fig. 2A for 30 min at 37°C, after which tryptase activity was determined. Mean and SD values are shown (n = 3).

FIGURE 3.

Glycosaminoglycan dose-response effects on β2-protryptase activation by CTSB (A) and CTSL (B). Each CTS was incubated with β2-protryptase and various concentrations of heparin, chondroitin sulfate A, and chondroitin sulfate B in sodium acetate buffer as for Fig. 2A for 30 min at 37°C, after which tryptase activity was determined. Mean and SD values are shown (n = 3).

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Of further interest, if β-protryptase had been incubated at acidic pH for 30 min at 37°C in the absence of heparin with CTSC, CTSB, or CTSL, and then with heparin, tryptase activity quickly appeared if CTSC had been used, but no activity was observed if either CTSL or CTSB had been used (data not shown). In the case of CTSC, this reflects formation of mature β-tryptase monomers and active tetramers only after heparin had been added. Why incubation of β-protryptase with CTSB or CTSL in the absence of heparin seemed to abrogate later activation was investigated by Western blotting. As shown in Fig. 4, β2-protryptase and β1/3-protryptase(s) in the absence of heparin were degraded by the endopeptidases, CTSL and CTSB. In contrast, CTSC, a dipeptidase, did not degrade β2-protryptase. Thus, heparin protects β-protryptases from degradation by CTSB and CTSL.

FIGURE 4.

Heparin protects β2 (A) and β1/3 (B) protryptases from degradation by CTSB and CTSL. Recombinant β2-protryptase was incubated with CTSL, CTSB, and CTSC, with or without heparin (A), and HMC-1 β1/3-protryptase with CTSL and CTSB, with or without heparin (B), for 30 min at 37°C. Portions of each incubation mixture were removed and subjected to Western blotting using G3 anti-tryptase mAb. Molecular mass marker positions are shown in kilodaltons.

FIGURE 4.

Heparin protects β2 (A) and β1/3 (B) protryptases from degradation by CTSB and CTSL. Recombinant β2-protryptase was incubated with CTSL, CTSB, and CTSC, with or without heparin (A), and HMC-1 β1/3-protryptase with CTSL and CTSB, with or without heparin (B), for 30 min at 37°C. Portions of each incubation mixture were removed and subjected to Western blotting using G3 anti-tryptase mAb. Molecular mass marker positions are shown in kilodaltons.

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The time courses for activation of β2-protryptase were similar for CTSL and CTSB. β-Protryptase (0.5 μg) was incubated with CTSL or CTSB (0.25 μg) in 100 μl 50 mM sodium acetate pH 6 for CTSL or pH 5.5 for CTSB, containing 25 μg heparin/ml, 150 mM NaCl, 1 mM EDTA, 5 mM l-cysteine, and 5% glycerol. The calculated t½ values to maximal activation for CTSL and CTSB were 4 and 5 min, respectively (Fig. 5A). To characterize further the processing of β-protryptase by CTSB and CTSL, Fig. 5B illustrates the dose-response relationship of these key components. Maximal generation of tryptase activity occurred with a 1:1 or greater weight ratio of each CTS to β-protryptase, which calculates to a molar ratio near unity. This nearly stoichiometric ratio has been reported previously for the processing of protrypsin by CTSB (32) and limits the amount of proenzyme that it can activate. The effect of ionic strength on β-protryptase processing by CTSB and CTSL revealed a <15% decline from 150 to 300 mM NaCl, a 30–35% decline at 500 mM, and an 80–90% decline at 1000 mM, which is similar to the effect of salt on the reactivation of inactive β-tryptase monomers to active tetramers at acidic pH (31). The optimal pH for processing of β-protryptase by CTSB and CTSL, as shown in Fig. 5C, is from 5 to 6. Again, this is comparable with the optimal pH for spontaneous conversion of inactive β-tryptase monomers to active β-tryptase tetramers. It is also similar to the pH optima determined for CTSL and CTSB with synthetic substrates Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, respectively (data not shown). Thus, the pH optima for CTSB and CTSL are similar to the optimal pH for converting β-tryptase monomers to tetramers.

FIGURE 5.

Physicochemical characteristics of β2-protryptase activation by CTSB and CTSL. A and B, Time course for activation of β2-protryptase. At the indicated times, one portion of the reaction mixture was removed for assessing tryptase activity with TGPK (A), and another portion was mixed with sample buffer for zymography (see Fig. 6B). B, Dose-response of CTSL and CTSB on β2-protryptase processing. Various concentrations of CTSL and CTSB were incubated with 5 μg/ml β-protryptase in 50 mM sodium acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM EDTA, 5 mM l-cysteine, and 5% glycerol for 30 min at 37°C and then assayed for tryptase activity. C, The pH dependence for activation of β2-protryptase by CTSL and CTSB. β2-Protryptase (5 μg/ml) was incubated with 2.5 μg/ml CTSL or CTSB in 50 mM sodium acetate buffer, pH 4–6.5, or 50 mM Tris buffer, pH 7–8.5, each containing 25 μg/ml heparin, 0.15 M NaCl, 1 mM EDTA, 5 mM l-cysteine, and 5% glycerol for 30 min at 37°C and then assayed for tryptase activity. Each point is the average of three determinations.

FIGURE 5.

Physicochemical characteristics of β2-protryptase activation by CTSB and CTSL. A and B, Time course for activation of β2-protryptase. At the indicated times, one portion of the reaction mixture was removed for assessing tryptase activity with TGPK (A), and another portion was mixed with sample buffer for zymography (see Fig. 6B). B, Dose-response of CTSL and CTSB on β2-protryptase processing. Various concentrations of CTSL and CTSB were incubated with 5 μg/ml β-protryptase in 50 mM sodium acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM EDTA, 5 mM l-cysteine, and 5% glycerol for 30 min at 37°C and then assayed for tryptase activity. C, The pH dependence for activation of β2-protryptase by CTSL and CTSB. β2-Protryptase (5 μg/ml) was incubated with 2.5 μg/ml CTSL or CTSB in 50 mM sodium acetate buffer, pH 4–6.5, or 50 mM Tris buffer, pH 7–8.5, each containing 25 μg/ml heparin, 0.15 M NaCl, 1 mM EDTA, 5 mM l-cysteine, and 5% glycerol for 30 min at 37°C and then assayed for tryptase activity. Each point is the average of three determinations.

Close modal
FIGURE 6.

CTSL and CTSB process β2-protryptase to enzymatically active tetramers based on gel zymography. A, Gelatin zymography. β2-Protryptase (β2-proT) and human lung tryptase (HLT) were used as shown. The bright gelatin-cleared bands of β2-protryptase treated with CTSL/B and heparin show the same electrophoretic mobility as human lung tryptase tetramer. In the absence of heparin or of CTSL/B, the dark band corresponds with β2-protryptase monomer (βTM). B, Fluorescent substrate zymography. Samples generated in Fig. 3A were analyzed as in 1Materials and Methods. A diffuse band of fluorescent product corresponds with the electrophoretic mobility of tryptase tetramer depicted in A and shows increasing intensity over the 30-min incubation.

FIGURE 6.

CTSL and CTSB process β2-protryptase to enzymatically active tetramers based on gel zymography. A, Gelatin zymography. β2-Protryptase (β2-proT) and human lung tryptase (HLT) were used as shown. The bright gelatin-cleared bands of β2-protryptase treated with CTSL/B and heparin show the same electrophoretic mobility as human lung tryptase tetramer. In the absence of heparin or of CTSL/B, the dark band corresponds with β2-protryptase monomer (βTM). B, Fluorescent substrate zymography. Samples generated in Fig. 3A were analyzed as in 1Materials and Methods. A diffuse band of fluorescent product corresponds with the electrophoretic mobility of tryptase tetramer depicted in A and shows increasing intensity over the 30-min incubation.

Close modal

Activation of β-protryptase to active β-tryptase tetramers could also be demonstrated by zymography. Gelatin zymography, as shown in Fig. 6A, shows clear separation of lung β-tryptase tetramer (right lane, clear band, βTT) from β2-protryptase monomer (left lane, dark band, βTM). The clear band signals proteolysis of gelatin resulting in less Coomassie blue staining, whereas the dark band reflects Coomassie blue staining of tryptase on a background of gelatin. CTSL and CTSB incubated with β2-protryptase in the absence of heparin (lanes2 and 3, respectively) failed to convert protryptase monomers to active tryptase tetramers, whereas these CTSs in the presence of heparin (lanes4 and 5, respectively) generated proteolytic activity that comigrated with tryptase tetramer. In Fig. 6B, fluorescent substrate zymography shows the generation of a band of peptidolytic activity after SDS-PAGE of an incubation mixture of CTSL (upper panel) and CTSB (lower panel), β2-protryptase, and heparin at acidic pH. This single somewhat diffuse band increased in intensity over a 30-min incubation time course and is observed where tryptase tetramers are expected to migrate based on Fig. 6A. The active β2-tryptase generated by CTSB and CTSL also behaved as a tetramer during heparin chromatography at pH 6, eluting at ∼0.5 M NaCl, whereas β2-protryptase eluted at ∼0.2 M NaCl. Finally, the N-terminal amino acid sequences of CTSL- and CTSB-generated β2-tryptase tetramers were analyzed by Edman degradation and consisted of IVGGQE, confirming that mature β-tryptase had been generated. Thus, both CTSB and CTSL serve in vitro as enzymes that process β-protryptase monomers to mature β-tryptase tetramers.

The CTSC pathway was previously demonstrated to involve autocatalytic cleavage at R−3 followed by removal of the pro′ dipeptides (3). The autocatalytic step appeared to be rate limiting. Consequently, a sigmoid time course for the two-step activation of β-protryptase and a more rapid exponential time course for the one-step activation of β-pro′tryptase were observed. Fig. 7A confirms the exponential and sigmoid CTSC activation curves (right panel) for β-pro′tryptase (closed circles) and β-protryptase (open circles), respectively. However, both β-protryptase and β-pro′tryptase exhibited rapid exponential time courses when activated with either CTSB or CTSL (Fig. 7A), suggesting that these CTSs might cleave β-protryptase directly between the −1/1 aa. This hypothesis was first tested with α-protryptase, which is not autoprocessed because Q rather than R resides in the −3 position (3), and perhaps because of the minimal proteolytic and peptidolytic activities of even mature tetrameric α-tryptase (10, 13). As shown in Fig. 7B, α-protryptase in the presence of heparin was activated by CTSB and CTSL, but not by CTSC, even though the sp. act. of mature α-tryptase with TGPK was ∼300-fold lower, as expected, than for β-tryptase. To test further the direct cleavage hypothesis, an R−3Q β-protryptase mutant was prepared and tested (Fig. 7C). This mutant should be resistant to autoprocessing because tryptic enzymes such as β-tryptase do not cleave next to Q. Although R−3Q β-protryptase was not activated by CTSC, both CTSB and CTSL produced fully active β-tryptase. Thus, CTSB and CTSL remove the entire propeptide from β-protryptase in the presence of heparin and thereby bypass the autoprocessing step.

FIGURE 7.

Mechanism for conversion of protryptase to mature tryptase by CTSL and CTSB. A, Comparative time courses for activation of pro versus pro′ β2-tryptases by CTSL, CTSB, and CTSC. Each CTS was added to pro or pro′ forms of β2-tryptase, and resultant tryptase activity was assessed with TGPK at various time points. B, Conversion of α-protryptase to α-tryptase by CTSL and CTSB, but not by CTSC. α-Protryptase (5 μg) was incubated with 5 μg CTSL, CTSB, or CTSC at 37°C for 30 min in a final volume of 200 μl sodium acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM EDTA, 50 μg/ml heparin, 5 mM l-cysteine, and 5% glycerol and assessed for tryptase activity with TGPK. C, Conversion of R−3Q β-protryptase to β-tryptase by CTSL and CTSB, but not by CTSC. R−3Q β-Protryptase (5 μg) was incubated with 5 μg CTSL, CTSB, or CTSC as in B and assessed for tryptase activity with TGPK. Average ± SD is shown from three independent experiments for each data point.

FIGURE 7.

Mechanism for conversion of protryptase to mature tryptase by CTSL and CTSB. A, Comparative time courses for activation of pro versus pro′ β2-tryptases by CTSL, CTSB, and CTSC. Each CTS was added to pro or pro′ forms of β2-tryptase, and resultant tryptase activity was assessed with TGPK at various time points. B, Conversion of α-protryptase to α-tryptase by CTSL and CTSB, but not by CTSC. α-Protryptase (5 μg) was incubated with 5 μg CTSL, CTSB, or CTSC at 37°C for 30 min in a final volume of 200 μl sodium acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM EDTA, 50 μg/ml heparin, 5 mM l-cysteine, and 5% glycerol and assessed for tryptase activity with TGPK. C, Conversion of R−3Q β-protryptase to β-tryptase by CTSL and CTSB, but not by CTSC. R−3Q β-Protryptase (5 μg) was incubated with 5 μg CTSL, CTSB, or CTSC as in B and assessed for tryptase activity with TGPK. Average ± SD is shown from three independent experiments for each data point.

Close modal

Activation of monomeric α- and β-protryptases were further examined by Superose 12 gel filtration and Western blotting for tryptase as shown in Fig. 8. Gel filtration was performed in 1.0 M NaCl to dissociate heparin from tryptase while maintaining the existing monomeric or tetrameric tryptase conformation. α-Protryptase by itself (Fig. 8A) eluted at a position with an apparent molecular mass of ∼30,000 Da, reflecting its monomeric quaternary conformation; CTSB and CTSC by themselves exhibited similar elution patterns, consistent with their molecular masses of ∼30,000 Da. α-Protryptase treated with either CTSB or CTSL in the presence of heparin at acidic pH was largely converted to tetrameric mature α-tryptase. This is reflected by most but not all of the CTSB-treated α-protryptase eluting with an apparent molecular mass of ∼130,000 Da, consistent with a tetrameric quaternary conformation of α-tryptase. The second protein peak eluting between 14 and 15 min consists of a mixture of CTSB and monomeric α-(pro)tryptase. In the second chromatogram, essentially all of the CTSL-treated α-protryptase eluted as a tetramer. In this case, the second protein peak consists of CTSL.

FIGURE 8.

Conversion of α-protryptase (A) and β2-protryptase (B) monomers to tryptase tetramers by CTSL and CTSB. α-Protryptase and β2-protryptase aliquots treated with CTSL or CTSB as above at acidic pH in the presence of heparin were subjected to Superose 12HR gel filtration chromatography in 50 mM NaCH3CO2, pH 6, containing 1.0 M NaCl, at a flow rate of 1 ml/min. Molecular mass markers from left to right at 669, 443, 200, 150, 66, and 29 kDa are indicated by downward solid triangles. Protein elution profiles were monitored by absorbance at a wavelength of 280 nm, and collected fractions (1 ml) were concentrated by ultrafiltration and Western blotted with G3 anti-tryptase mAb to detect tryptase. In B, tryptase activity was monitored with TGPK.

FIGURE 8.

Conversion of α-protryptase (A) and β2-protryptase (B) monomers to tryptase tetramers by CTSL and CTSB. α-Protryptase and β2-protryptase aliquots treated with CTSL or CTSB as above at acidic pH in the presence of heparin were subjected to Superose 12HR gel filtration chromatography in 50 mM NaCH3CO2, pH 6, containing 1.0 M NaCl, at a flow rate of 1 ml/min. Molecular mass markers from left to right at 669, 443, 200, 150, 66, and 29 kDa are indicated by downward solid triangles. Protein elution profiles were monitored by absorbance at a wavelength of 280 nm, and collected fractions (1 ml) were concentrated by ultrafiltration and Western blotted with G3 anti-tryptase mAb to detect tryptase. In B, tryptase activity was monitored with TGPK.

Close modal

Fig. 8B shows the Superose 12 elution profiles and Western blots for β2-protryptase treated as above. β2-Protryptase by itself eluted with an apparent molecular mass of ∼30,000 Da, consistent with it being a monomer. CTSB (top panel) and CTSL (middle panel) converted essentially all of the β2-protryptase to a tetramer based on its elution near the 150,000 Da molecular mass marker. In addition, tryptase activity was detected in association with tryptase tetramers generated by CTLB and CTSL. Thus, based on generation of tryptase enzymatic activity and conversion of protryptase monomers to tetramers, CTSB and CTSL (at acidic pH in the presence of heparin) each directly activates α-protryptase, β-protryptase, and β-pro′tryptase (Fig. 9).

FIGURE 9.

Pathways for conversion of α/β-protryptases to their mature counterparts. β2-Protryptase but not α-protryptase can undergo autocatalytic cleavage to pro′tryptase due to Arg in the −3 position. Both α- and β-protryptases can be processed directly to their mature counterparts by CTSB and CTSL in the absence of autocatalytic processing.

FIGURE 9.

Pathways for conversion of α/β-protryptases to their mature counterparts. β2-Protryptase but not α-protryptase can undergo autocatalytic cleavage to pro′tryptase due to Arg in the −3 position. Both α- and β-protryptases can be processed directly to their mature counterparts by CTSB and CTSL in the absence of autocatalytic processing.

Close modal

The current study identifies CTSB and CTSL as two processing proteases for human α/β-protryptases (Fig. 9). CTSC, based on murine studies, is not critical for protryptase processing, at least in mice. The involvement of CTSB and CTSL in the processing of human β-protryptases was first suspected when these two enzymes were identified by mass spectroscopy in partially purified preparations of protein(s) from HMC-1 cells that had been chromatographically separated from CTSC but nevertheless exhibited β-protryptase processing activity. That these CTSs could indeed process β-protryptase to maturity was then demonstrated with purified enzymes and validated with specific inhibitors. The optimal pH for processing, 5.5–6, coincided with the pH optima of both CTSs for cleaving synthetic substrates and for the spontaneous reactivation of mature tryptase monomers to tetramers. Also, heparin was required to obtain active β-tryptase tetramer and to protect (pro)tryptase from degradation by CTSB and CTSL. The importance of this latter observation might have been reflected in a study showing that heparin-deficient murine mast cells have lesser amounts of tryptase than those from wild-type mice; that is, CTSB, CTSL, and possibly other proteases may have degraded tryptase that was not protected by heparin (33).

CTSL and CTSB have been linked to the processing of proenzymes or other proteins in a variety of different cell types (3436). In chromaffin cells, CTSL processes proenkephalin to [Met]enkephalin (36). In pancreatic exocrine cells, CTSB activates trypsinogen to trypsin (35). CTSL and CTSS but not CTSB process proCTSC to its active tetrameric form (37). CTSL processes invariant chain (Ii) in cortical thymic epithelial cells (38), and the p41 alternative splice variant of Ii binds to and inhibits CTSL (39). CTSL processes Ag in cortical thymic epithelial cells and thereby influences positive selection of developing CD4+ lymphocytes (40). CTSL deficiency introduced into NOD mice diminishes production of CD4 T cells and attenuates the development of autoimmune diabetes (41). Thymocyte expression of CTSL also is critical for the development of NKT cells, perhaps through processing of putative natural CD1d ligands needed for NKT cell selection (42).

In the current study, CTSB and CTSL processed β-protryptase to maturity with no delay, unlike the autocatalytic–CTSC pathway where rate-limiting autocatalysis results in a sigmoid time course. This suggested that these CTSs directly process β-protryptase in the absence of β-protryptase autoprocessing. To test directly this hypothesis, R−3Q β-protryptase was prepared. This construct did not undergo autoprocessing and thus was not processed to maturity by the autocatalytic–CTSC pathway. However, both CTSB and CTSL, in the presence of heparin at acidic pH, processed R−3Q β-protryptase to mature, fully active β-tryptase tetramers as observed by gel filtration and zymography under nondenaturing conditions and by N-terminal amino acid sequence analysis.

Direct removal of the propeptide from β-protryptase by CTSB and CTSL raised the possibility that α-protryptase might also be processed by CTSB and CTSL. A mechanism by which α-protryptase could be activated has not been reported. The current study shows that recombinant α-protryptase was processed by these CTSs to maturity as reflected by formation of tetramers in the presence of heparin with, as expected, only modest peptidolytic activity (10, 13). However, it appears unlikely that a large portion of the tryptase stored in mast cell secretory granules is α-tryptase, because the sp. act. of tryptase purified from human mast cells is comparable with that of recombinant β-tryptase. Another possibility is that αβ-heterotetramers could form. That different tryptases can form heterotetramers has been demonstrated in the murine system with MMCP-6 and MMCP-7 (43). Also notable is that protryptases with two N-linked carbohydrates (recombinant α- and HMC-1 β1/3-protryptases) as well as β2-protryptase with one N-linked carbohydrate can be converted to their mature forms by CTSL and CTSB.

Why R is conserved at the −3 position of protryptases in most species is unclear. A study of tryptase evolution found that the R−3Q change is a recent event, detected in humans but not chimpanzees or lower primates (44). This substitution also is found in human δ-protryptase (45), a form of tryptase with an α-tryptase-like G235D, but unlike α/β-tryptases it also contains a stop codon 40 aa shy of the expected C terminus. An R−3E mutation has been reported in gerbil protryptase (46), which would likely be susceptible to direct processing by CTSB and CTSL, but not by the autocatalytic–CTSC pathway. Furthermore, mastins, proteases with homology to tryptases, have propeptides without a basic residue at the −3 position yet are processed to active multimeric proteases and then stored in secretory granules (47). Protryptases and homologous zymogens lacking R−3 do not require autocatalysis for processing and might be processed exclusively through CTSB/CTSL-like pathways. Such redundancy for processing protryptases might be important to mast cells, because the cell devotes a considerable amount of energy to generating large quantities of tryptase(s) (48).

In summary, CTSL and CTSB are novel processing enzymes for human β-protryptases and also for human α-protryptase.

This work was supported in part by National Institutes of Health Grants R01-AI27517 and U19-AI77435 (to L.B.S.).

Abbreviations used in this article:

CM

carboxymethyl

CTS

cathepsin

HMC-1

human mast cell leukemia cell-1

MCA

7-amido-4-methylcoumarin substrate

MMCP

murine mast cell protease

SP

sulfopropyl

TGPK

tosyl-l-Gly-Pro-Lys-p-nitroanilide.

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L.B.S. receives royalties from Virginia Commonwealth University that have been collected from Phadia for their commercial tryptase assay.