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Characterization of cathepsin L secreted by Sf21 insect cells
ABB Archives of Biochemistry and Biophysics 444 (2005) 7–14 www.elsevier.com/locate/yabbi
Characterization of cathepsin L secreted by Sf21 insect cells Gary D. Johnson *, Weiping Jiang R&D Systems, Inc. 614 McKinley Pl. NE, Minneapolis, MN 55413, USA Received 26 July 2005, and in revised form 20 September 2005 Available online 21 October 2005
Abstract Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni2+ affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed. 2005 Elsevier Inc. All rights reserved. Keywords: Cysteine proteases; Cathepsin L; Subsite specificity; Cystatins; Monoclonal antibodies
The majority of known cathepsins are peptidases that utilize a catalytic cysteine residue in their active site. These enzymes, structurally related to papain, belong to the C1 peptidase family [1]. While C1 cathepsins are generally endopeptidases, there are known examples of exopeptidases in the family (e.g., cathepsins C and X) [2]. Most cathepsins are present in lysosomes, where they play a major role in intracellular protein degradation [1,2]. Cathepsins are also present in other cellular compartments, or are secreted and associated with the plasma membrane, allowing them to perform additional functions. Cathepsins are known to be involved in antigen presentation [1,3,4], zymogen activation [5], and prohormone processing [6]. Most cathepsins are ubiquitously expressed, but others have a more restricted tissue distribution [1,2], resulting in specialized functions. For example, cathepsin K is secreted by osteoblasts and plays a major role in bone resorption. Cathepsin K deficiencies are responsible for the bone diseases pycnodysostosis and osteopetrosis [7,8].
*
Corresponding author. E-mail address: [email protected] (G.D. Johnson).
0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.09.011
Cathepsins of the C1 family are expressed by most multicellular eukaryotes, including insects. The majority of insect cathepsins are homologous to mammalian cathepsins B and L [9]. A number of secreted insect cathepsins may be involved in the protein degradation that occurs during food digestion [10], embryogenesis [11,12], and metamorphosis [13,14]. Insect cathepsins may also be a part of a defense system, eliminating foreign proteins by degradation [15]. Sf21 insect cells are derived from pupal ovarian cells of Spodoptera frugiperda (Fall Armyworm) [16]. These cells are frequently used for the heterologous expression of recombinant proteins. During several attempts at the expression of mammalian cathepsins in the Sf21 cell line, we observed that the cell line secreted a protease with properties similar to mammalian cathepsins that also co-purified with the human enzymes. The insect protease was purified and biochemically characterized. The pH/activity and inhibitor profiles showed that the insect protease is a cysteine protease of the C1 family. Substrate specificity studies and amino acid sequence data revealed that the protease is an insect homologue of mammalian cathepsin L. Monoclonal antibodies were raised against the pro and
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mature forms of the insect cathepsin L. These antibodies are useful for the detection of the enzyme, and possibly its purification by immunoaffinity methods. Materials and methods Protein purification All purification steps were performed at 6–10 C. Sf21 cells were grown in Ex-cell 401 serum-free insect medium from JRH Biosciences (Lenexa, KS) to a density of 1.4 · 107 cells/ml. Conditioned medium was harvested, concentrated 10-fold by tangential flow ultrafiltration, and the pH was adjusted to 7.5 with 1 N NaOH. Insoluble material was removed by centrifugation at 21,000g. The soluble protein was diluted 4-fold with cold 5 mM Hepes,1 0.1 M NaCl, pH 7.5, then loaded onto an SP Sepharose (GE Healthcare, Piscataway, NJ) column equilibrated in 25 mM Hepes, 0.1 M NaCl, pH 7.5 (buffer A). Bound proteins were eluted using a linear gradient of NaCl (0.1–1 M) in buffer A, with the fractions collected in tubes containing 1 M NaCl to minimize precipitation of the eluted protein. Fractions were assayed, after autoactivation, using the substrate benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide (Z-FR-MCA) at 10 lM in 50 mM NaOAc, 1 mM DTT, pH 5.0. The fractions with high specific activity were pooled. A 5-ml chelating Sepharose column (GE Healthcare) was charged with Ni2+ ions, then equilibrated in 25 mM Hepes, 0.5 M NaCl, and 10 mM imidazole, pH 7.5 (buffer B). Protein pooled from the SP Sepharose column was diluted 4-fold in buffer B and loaded onto the chelating Sepharose column. The column was washed with buffer B before the elution of bound proteins with 0.2 M imidazole, 0.5 M NaCl, pH 7.5. The protein eluted from the chelating Sepharose column was concentrated to 4 ml by ultrafiltration using a 30-kDa NMWL Ultracel membrane in an Amicon stirred cell from Millipore (Billerica, MA), and insoluble material removed by centrifugation at 16,000g. The concentrated soluble protein was then subjected to gel permeation chromatography on a 2.5 · 90 cm Superdex 200 (GE Healthcare) column equilibrated in 25 mM Hepes, 0.5 M NaCl, and 0.1% Brij 35, pH 7.5. The Superdex 200 column was calibrated with molecular mass standards (ferritin, catalase, aldolase, albumin, ovalbumin, and chymotrypsinogen A) purchased from GE Healthcare to determine the molecular mass of the native enzyme. SDS–PAGE was performed using Tris–glycine gels with molecular mass standards purchased from BioRad Laboratories (Hercules, CA).
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Abbreviations used: Boc, tert-butoxycarbonyl; DTT, dithiothreitol; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; Hepes, N-(2-hydroxyethyl)piperazine-N 0 -(2-ethanesulfonic acid); MCA, 4-methyl-7-coumarylamide; LC–MS/MS, liquid chromatography nano-spray mass spectrometry/mass spectrometry; Mes, 2-(N-morpholino)ethanesulfonic acid; NaOAc, sodium acetate; RFU, relative fluorescence unit; Z, benzyloxycarbonyl.
Amino acid sequence analysis The purified enzyme in solution was submitted to the Molecular Structure Facility of the University of California at Davis for NH2-terminal sequence analysis by the Edman degradation procedure. The resulting amino acid sequence was used to search protein databanks for sequence matches using the Blastp program [17]. The selected sequence matches were aligned using Clustal W [18]. The purified protein was also submitted to the Molecular Biology and Biophysics Proteomic Analysis Core facility at the University of Minnesota, St. Paul for internal peptide analysis involving in-gel trypsin digestion followed by liquid chromatography nano-spray mass spectrometry/ mass spectrometry (LC–MS/MS). Determination of carbohydrate content The carbohydrate content of the purified Sf21 protease was measured using a glycoprotein carbohydrate estimation kit purchased from Pierce Biotechnology (Rockford, IL). The Sf21 protease and standard proteins of known carbohydrate content were first oxidized by sodium metaperiodate, then the resulting aldehydes were reacted with an aldehyde detection reagent as recommended by the manufacturer. The absorbance at 550 nm was then measured for the standard proteins and the Sf21 protease, and a standard curve was generated to relate the absorbance to percentage carbohydrate content. Lysozyme was used as a negative control. Positive controls were ovalbumin, apotransferrin, fetuin, and a1-acid glycoprotein. Enzyme autoactivation and assay optimization To determine the optimal conditions for autoactivation of the cathepsin, purified cathepsin was diluted >20-fold (to 0.02 mg/ml) in 0.1 M NaOAc, and 10 mM DTT, pH 5.0, then incubated at 37 C to permit autoactivation. At various time points aliquots of the incubation mixture were removed for immediate assay using the Z-FR-MCA substrate. At each time point, 2 lg aliquots of protein were removed and the protein was precipitated by the addition of trichloroacetic acid to a final concentration of 10%. The precipitated protein was saved for analysis by SDS– PAGE under reducing conditions. The activity of the Sf21 protease was examined under conditions in which the pH and ionic strength of the reaction buffer were varied. Purified enzyme autoactivated in 0.1 M NaOAc, 10 mM DTT, pH 5.0, was diluted 20-fold in 10 mM DTT, then diluted an additional 10-fold into microplate wells containing NaOAc buffers (pH 3–5) or Mes buffers (pH 5.5–7). Buffer concentrations were 55 mM, and the final DTT concentration was 1 mM. NaCl concentration was varied at each pH from 0–1 M. Reactions were initiated by the addition of Z-FR-MCA substrate to 10 lM final concentration. Reaction rates were monitored by measuring the rates of liberation of free 7-
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amino-4-methylcoumarin on a SpectraMax Gemini fluorescence plate reader from Molecular Devices (Sunnyvale, CA) using the excitation and emission wavelengths of 380 and 460 nm, respectively. Substrate specificity of the Sf21 cathepsin Z-LR-MCA and Z-FR-MCA substrates were obtained from R&D Systems (Minneapolis, MN). All other peptidyl-MCA substrates were purchased from Bachem (Torrance, CA). Peptidyl-MCA substrates were used at 10 lM in a buffer consisting of 50 mM NaOAc, 1 mM DTT, pH 5.0. Reactions were started by the addition of activated enzyme to microplate wells, and the resulting fluorescence increases were recorded using a fluorescence plate reader at 24 C. Relative fluorescence units (RFU) were converted to moles of product using a conversion factor generated using purified 7-amino-4-methylcoumarin from Sigma (St. Louis, MO) as the standard. Azocasein (Sigma) degradation assays were performed as described by Wolz and Bond [19], except that 50 mM Mes, 2.5 mM DTT, pH 5.0 was used as the assay buffer.
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cells. Conditioned media samples from the resulting hybridomas were screened for binding to the Sf21 cathepsin by Western blot analysis. Sf21 conditioned medium was run on 12% Tris–glycine gels, the fractionated proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories), and incubated with hybridoma culture medium diluted in Tris-buffered saline containing 1% nonfat dried milk and 0.1% Tween 20. The immunoblots were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (R&D Systems) and developed using Western Glo chemiluminescent detection reagents (R&D Systems). Hybridoma culture media samples that reacted with the 48-kDa Sf21 protease band, but not with other Sf21 secreted proteins, were judged to be positive. Positive hybridoma clones were expanded and the secreted antibodies were purified by Protein G affinity chromatography. Immunoblot analysis of the purified monoclonal antibodies was used to identify antibodies specific for the pro and mature domains of the Sf21 cathepsin. Results and discussion Enzyme purification
Determination of kinetic constants KM and Vmax values for cathepsin hydrolysis of Z-FRMCA were determined using a concentration of 0.484 nM of enzyme in a buffer composed of 50 mM NaOAc, 1 mM DTT, pH 5.0, at 24 C. Substrate concentrations were varied from 2 to 100 lM. Initial rate data were recorded as RFU s1 using a fluorescence plate reader. Conversion factors for RFU to moles of product were determined for each substrate concentration to correct for inner filter effects. The corrected initial rate data were fit to the Michaelis–Menten equation by nonlinear regression analysis using Kaleidagraph software from Synergy Software (Reading, PA). The value of kcat was calculated from Vmax using the enzyme concentration determined by active site titration with the inhibitor E-64 as described by Knight [20]. Recombinant human cystatins (A, B, C, D, E/M, F, S, SA, and SN) were obtained from R&D Systems. Ki values for the cystatins were determined using a constant concentration of 0.97 nM of activated cathepsin, and cystatins were varied 20–60-fold in concentration. Enzyme and inhibitor were preincubated for 30 min at 24 C before the addition of Z-FR-MCA substrate. The initial reaction rates were measured at three substrate concentrations (5, 10, and 25 lM) for each inhibitor concentration using a fluorescence plate reader. The resulting data were fit to a model for competitive inhibition (v = Vmax)/(1 + KM/ [S] + (1 + [I]/Ki)) using a computer program developed by Hernandez and Ruiz [21]. Monoclonal antibody production and characterization B cells obtained from mice immunized with purified Sf21 secreted cathepsin were fused with mouse myeloma
The Sf21 protease was purified to electrophoretic homogeneity using ion-exchange chromatography (SP Sepharose), immobilized metal affinity chromatography (chelating Sepharose), and gel-permeation chromatography (Superdex 200). All purification steps were carried out at pH 7.5 to prevent the autoactivation and subsequent degradation of the enzyme that occurs at acidic pH. SDS– PAGE analysis of the purification pools under reducing conditions is shown in Fig. 1. The purified protease has
Fig. 1. SDS–PAGE analysis of Sf21 protease purification. Lane 1, conditioned medium; lane 2, SP Sepharose pool; lane 3, chelating Sepharose pool; and lane 4, Superdex 200 pool. Lane M contains molecular mass standards. Molecular masses of the standards (kDa) are displayed on the right. Proteins were visualized by silver staining.
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Table 1 Purification of Sf21 protease from 10 L of conditioned medium Pool
Total protein (mg)
Total activity (lmol min1)
Specific activity (lmol mg1 min1)
Purification factor
Yield (%)
Conditioned medium SP Sepharose Chelating Sepharose Superdex 200
1620 54 31 15.8
510 160 200 160
0.32 3.0 6.6 10.1
1 9.6 20.8 32
100 31 39 31
an apparent subunit molecular mass of 48 kDa. From lane 1, it is evident that the enzyme is one of the more abundant proteins secreted by the Sf21 cells, as the 48 kDa protein band is clearly visible in the mixture of proteins in the conditioned medium. The protease is substantially purified by the SP Sepharose step (lane 2), with the remaining contaminants removed by the chelating Sepharose and Superdex 200 steps (lanes 3 and 4). The results of a typical purification are summarized in Table 1. Activity measurements of several batches of the conditioned medium using the Z-FR-MCA substrate indicate that the enzyme is expressed at a level of 5 mg/L or greater. At least half of this activity was generally lost during the SP Sepharose step due to the tendency of the enzyme to precipitate under conditions of low ionic strength at neutral pH. After the SP Sepharose step, the inclusion of 0.5 M NaCl in all buffers minimized precipitation of the enzyme. Typically, a yield of 1.5–2.0 mg of purified enzyme per liter of conditioned medium was achieved. The activity of the purified protease was completely inhibited by E-64, but no inhibition was observed using either the serine protease inhibitor 3,4-dichloroisocoumarin or the aspartic protease inhibitor Pepstatin A (data not shown), evidence that the enzyme is a member of the C1 cysteine peptidase family. The subunit molecular mass of the Sf21 protease was estimated to be 48 kDa by SDS–PAGE, but the enzyme behaved as a much larger protein during the gel permeation chromatography step. The Superdex 200 column used for purification of the enzyme was calibrated with protein molecular mass standards. From these data, a molecular mass of 405 kDa was calculated for the native Sf21 secreted protease. Therefore, the protease is oligomeric, having a mass expected for an octamer. Amino acid sequence analysis of the purified Sf21 secreted protease
the cotton bollworm cathepsin L peptides with corresponding masses, and that the two proteins share a very high degree of amino acid sequence identity. Fig. 2 displays an alignment of the four Sf21 protease peptides with the amino acid sequences of procathepsin L from cotton bollworm, fruit fly, and human. The NH2-terminal sequence of the Sf21 protease, located at the beginning of the propeptide, is very similar to the insect sequences, but has little identity with the human sequence. However, the tryptic peptides, all located within the catalytic domain, display a much greater degree of interspecies identity. These peptides are 100, 68, and 76% identical to the corresponding fruit fly sequences, and 58, 53, and 59% identical to the corresponding human procathepsin L sequences. This high degree of sequence identity is consistent with the hypothesis that the Sf21 secreted protease is an insect cathepsin L. Assuming 100% sequence identity with the cotton bollworm protein, the Sf21 protein is initially synthesized as a 341 amino acid preproenzyme, becoming a 325 residue zymogen after removal of a 16 residue signal sequence. By analogy with human cathepsin L, generation of the mature, active Sf21 cathepsin L would require the removal of a 110–115 residue propeptide [22]. Determination of carbohydrate content The predicted molecular mass of the Sf21 cathepsin zymogen is approximately 35 kDa. However, the subunit molecular mass was estimated to be 48 kDa by SDS– PAGE, indicating that the protein may be glycosylated. The presence of carbohydrate modifications on the protease was confirmed using periodate oxidation of the purified cathepsin, followed by detection of the resulting aldehydes. From a comparison with standard proteins of known carbohydrate content, it was estimated that the Sf21 cathepsin zymogen contains >25% carbohydrate. Autoactivation of the purified Sf21 cathepsin L
The NH2-terminal sequence of the Sf21 secreted protease zymogen was determined. The NH2-terminal sequence (VSLLDLVRE) is identical to the NH2-terminal sequence of a putative procathepsin L from the cotton bollworm as deduced from its cDNA (GenBank Accession No. AAQ75437). The molecular masses of three tryptic peptides derived from the Sf21 protease, a total of 48 amino acid residues, were determined to be the same as three deduced tryptic peptides of the cotton bollworm cathepsin L. Therefore, it is probable that these three tryptic peptides of the Sf21 protease are identical in amino acid sequence to
Preliminary tests revealed that the Sf21 cathepsin L remains stable as the zymogen at pH 7.5, but undergoes autoactivation at pH 5 or lower, and that complete activation required the presence of a reducing agent such as DTT. SDS–PAGE analysis of the time course for activation under the conditions described is shown in Fig. 3. Aliquots at each time point were also monitored for activity using the substrate Z-FR-MCA (data not shown). The purified enzyme is initially present as the 48-kDa proform, which has only 2% of the activity of the fully activated
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Fig. 2. Amino acid sequence alignments of procathepsin L from Sf21, cotton bollworm (cb, AAQ75437), fruit fly (ff, Q95029), and human (h, P07711). Identical and similar residues are indicated by * and :, respectively. The sequences underlined in cb are perfect matches to the sequences determined for the Sf21 protease. The NH2-terminal sequence of the Sf21 proenzyme was determined from the intact protein in solution. The masses (Mr experimental/ calculated) listed above the three internal peptides were the results from LC–MS/MS analysis of tryptic peptides. The Cys residue in the last peptide was modified by a carbamidomethyl group.
Fig. 3. Autoactivation of the Sf21 cathepsin at pH 5.0. The numbers above each lane indicate the time point in minutes at which aliquots were removed from the autoactivation reaction for SDS–PAGE analysis. Molecular mass standards were run in lane M, with molecular masses of the standards (kDa) shown on the left.
enzyme. Activation proceeded slowly for the first 20 min, until the proform was partially converted to a 34-kDa species. Within 30 min, much of the proform was converted to the 34-kDa form, and a 32-kDa form was also beginning to appear. Processing was complete by 40 min of incubation, with the conversion of the proform to the 32 kDa mature
enzyme form. From activity measurements it was apparent that both the 34- and 32-kDa enzyme forms are highly active (data not shown). Trace amounts of propeptidesized fragments (14 and 16 kDa) were visible at the 30and 40-min time points on overexposed gels, but were quickly degraded and no longer detected with silver staining by the 60-min time point. It is clear that the Sf21 protease proform efficiently transforms itself into the mature, active enzyme form under acidic conditions. Autocatalytic processing of the precursor to the mature enzyme form by removal of the NH2-terminal propeptide has also been reported for the cysteine endopeptidases papain [23] and cathepsin L [22] under similar conditions. By contrast, procathepsins S and B are able undergo partial activation to intermediate forms [23,24] which can be trimmed by exopeptidases to generate the mature enzyme form. Dependence of Sf21 cathepsin L activity on pH and ionic strength The common cathepsin substrate Z-FR-MCA was used to examine the effects of pH and ionic strength on the activity of the Sf21 cathepsin L. The resulting data are presented in the three-dimensional bar graph of Fig. 4, with
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G.D. Johnson, W. Jiang / Archives of Biochemistry and Biophysics 444 (2005) 7–14 Table 2 Rates of hydrolysis of peptidyl-MCA substrates by the secreted Sf21 cathepsin
Fig. 4. Effects of pH and NaCl concentration on Sf21 cathepsin hydrolysis of Z-FR-MCA. NaCl concentrations (M) are shown on the left axis and coded in the bar graph: black, 0 M; white, 0.1 M; diagonal stripes, 0.25 M; and stippled, 1.0 M. Buffer pH is shown on the center axis. Enzyme activity in relative fluorescence U/s (RFU/s) is displayed on the right axis.
activity displayed as relative fluorescence U/s (RFU/s). The enzyme is active in the pH range 3.0–6.5, with optimal activity at pH 5.0–6.0. pH/activity profiles have been determined for a number of cathepsins using the Z-FR-MCA substrate. Most of these enzymes were observed to be active in the pH range 3.5–8.5. These enzymes include cathepsins S [25], K [26,27], and B [28]. Cathepsin L, however, is known to be active in the pH range 3.0–6.5 [29]. The Sf21 cathepsin most resembles cathepsin L in its pH/ activity profile. Activity was greatest at low ionic strength, decreasing by about 40% at most pH values tested as NaCl was added to 0.1 and 0.25 M in the reactions. Raising (NaCl] from 0.25 to 1.0 M resulted in slight activity increases in the pH 4.0–5.0 range, but resulted in decreased activity at pH 3.0 and at pH 5.0 or higher. As a whole, the effect of ionic strength on the Sf21 cathepsin activity is modest near the optimal pH, but significant at low pH and near neutral pH. Subsite specificity mapping using peptidyl-MCA substrates Peptidyl-MCA substrates are commonly used to assay the activity of cysteine proteases of the C1 family. A variety of peptidyl-MCA substrates, all having a common P01 group (MCA), were used to probe the P1 and P2 subsite specificity of the Sf21 cathepsin (see [30] for a discussion of peptidase subsite notation). One advantage of using these substrates is the requirement that the 7-amino-4methylcoumarin (P01 ) group must be released in order for a fluorescence increase to occur, thereby defining the positions of P1 and P01 in the substrates. The peptidyl-MCA substrates were reacted with the insect cathepsin, and the rates are expressed as specific activities (lmol mg1 min1) in Table 2. The substrates used are ranked from highest to lowest specific activity in the table. The enzyme exhibits a clear preference for Arg residues in the P1 position, demon-
Substrate
Specific activity (lmol mg1 min1)
Z-FR-MCA Z-LR-MCA PFR-MCA succinyl-AFK-MCA LY-MCA Boc-RR-MCA Succinyl-LLVY-MCA Boc-RVRR-MCA Boc-VPR-MCA R-MCA GR-MCA Z-GGR-MCA Boc-IEGR-MCA Z-GP-MCA Succinyl-AAPF-MCA Z-AAN-MCA Acetyl-IEPD-MCA
11.0 8.7 3.9 0.38 0.36 0.03 0.012 0.010 0.008 ND ND ND ND ND ND ND ND
Conditions for hydrolysis are described in Materials and methods. The specific activity values reported are the average of three separate determinations. ND, no hydrolysis detected.
strated by the high rates of hydrolysis of Z-FR-MCA, ZLR-MCA, and PFR-MCA. Lys and the hydrophobic residue Tyr were also accepted at P1 (succinyl-AFK-MCA and LY-MCA), but these substrates were hydrolyzed at a rate at least an order of magnitude more slowly than the best substrates having Arg at P1. No hydrolysis was detected for the substrates having Pro, Asn, or Asp in the P1 position. Despite the preference for P1 Arg residues, no hydrolysis of Arg-MCA was detected, indicating that the enzyme is not an effective aminopeptidase. A comparison of the substrates with P1 Arg residues indicates a preference for large hydrophobic residues in the P2 position, as shown by the presence of Phe or Leu at P2 for all five of the substrates that were hydrolyzed at high rates. Furthermore, comparison of the hydrolysis rates of Z-FR-MCA and ZLR-MCA reveals a preference for Phe at P2. This pattern of subsite specificity is very similar to that of the mammalian cysteine proteases cathepsin B and cathepsin L [25,31]. These two cathepsins, however, differ in their hydrolysis of substrates with P2 Arg residues. Cathepsin B hydrolyzes P2 Arg substrates with rates comparable to P2 Phe and Leu substrates [32], but P2 Arg substrates are hydrolyzed relatively slowly by cathepsin L [26,32]. In the present survey of peptidyl-MCA substrates for the Sf21 secreted cysteine protease, it is clear that peptides with a P2 Arg residue (Boc-RR-MCA and Boc-RVRR-MCA) are cleaved at much lower rates than substrates with hydrophobic P2 residues. Therefore, the results of this study indicate that the Sf21 cathepsin is an endopeptidase with subsite specificity closely resembling that of cathepsin L. The values of KM and kcat were determined for Z-FRMCA, the best of the peptidyl-MCA substrates tested. The KM was determined to be 11 lM (average of five experiments, data not shown), a value typical for cathepsins of the C1 cysteine peptidase family, which generally fall in
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the 5–20 lM range [24–26]. The kcat/KM of 1.6 · 106 M1 s1 is intermediate to those reported for human cathepsins B and L [25,26]. Protease activity of the purified Sf21 cathepsin The hydrolysis of the protein substrate azocasein by the purified cathepsin was measured as described in Materials and methods. It was determined that 1 mg of the cathepsin is able to solubilize 23.7 mg azocasein min1 at 37 C. Therefore, the Sf21 cathepsin L is a very active protease as well as a peptidase. Cystatin inhibitors of the Sf21 cathepsin A panel of cystatins was tested for the inhibition of the Sf21 cathepsin. Initial tests revealed that human cystatins A, B, C, D, E/M, F, SA, and SN are all potent inhibitors of the enzyme, but that human cystatin S inhibits it only weakly. The equilibrium constant for dissociation of complexes (Ki) between the Sf21 cathepsin and various cystatins was determined for the more potent inhibitors, and the results are presented in Table 3. Although cystatin C appeared to have a Ki value in the range of 0.1–0.5 nM, a Ki for this inhibitor is not included in Table 3 because the data obtained were not a precise fit to the model for competitive inhibition. Of the cystatins for which accurate Ki values could be determined, cystatins A, B, E/M, F, SA, and SN were found to be subnanomolar inhibitors of the Sf21 cathepsin, with Ki values in the range of 0.19– 0.51 nM. These cystatins are as potent as E-64, the active site-directed inhibitor that is frequently used for the inhibition of papain-like cysteine proteases. We have determined a Ki value of 0.59 ± 0.13 nM for E-64 inhibition of the Sf21 cathepsin L (data not shown). Cystatin D, with a Ki of 6.5 nM, was somewhat less potent than the other cystatins included in Table 3. The Ki values for the inhibition of mammalian cathepsins B, H, L, and S by cystatins A, B, C, and D have been previously determined [33–38] and summarized by Abrahamson et al. [39]. A comparison of these data with the present study indicates that the Sf21 cathepsin exhibits an inhibitor profile closely resembling that of cathepsin H, for which Ki values of 0.31, 0.58, 0.28, and 7.5 nM have been determined for cystatins A,
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B, C, and D, respectively. The cystatin inhibition profile of the Sf21 cathepsin is also similar to those of cathepsins L and S, except for a less potent inhibition of the insect protease by cystatin C. The cystatin inhibitor profile of the Sf21 cathepsin is very distinct from that of cathepsin B. Specificity of monoclonal antibodies raised against the insect cathepsin The majority of the monoclonal antibodies raised against the insect cathepsin were found to recognize both the zymogen and the mature, activated enzyme as judged by Western blot analysis. However, one clone produced an antibody specific for the cathepsin propeptide. A comparison of the specificity of two of the monoclonal antibodies as assayed by immunoblotting is shown in Fig. 5. Primary antibodies MAb 1 (clone 193702) and MAb 2 (clone 193720) at 1 lg/ml were used to detect the Sf21 cathepsin zymogen and autoactivated mature enzyme. The purified zymogen was loaded in lanes 1 and 5 at 20 ng, and at 100 ng in lanes 2 and 6. Fully activated, mature cathepsin was loaded at 100 ng in lanes 3 and 7. Partially activated cathepsin was loaded at 400 ng in lanes 4 and 8. MAb 1 was used as the primary antibody for lanes 1-4, MAb 2 was the primary antibody for lanes 5–8. Both antibodies are able to detect as little as 20 ng of the 48-kDa zymogen, but MAb 1 is slightly more sensitive, as its signal in lane 1 is stronger than lane 5. Only MAb 1 is able to detect the 32 kDa mature enzyme, judging from the strong signal in lane 3 compared to the absence of a signal in lane 7. It is apparent that the epitope for MAb 2 lies partly or entirely in the cathepsin propeptide because of its failure to recognize the mature form of the enzyme. Comparing lanes 4 and 8, it is evident that MAb 1 recognizes several activation intermediates between 34 and 40 kDa that are
Table 3 Inhibition of the Sf21 secreted cathepsin by human cystatins Cystatin
Ki (nM) ± standard deviation
A B D E/M F SA SN
0.21 ± 0.026 0.46 ± 0.040 6.5 ± 1.5 0.19 ± 0.054 0.21 ± 0.025 0.51 ± 0.11 0.27 ± 0.021
Ki values were determined as described in Materials and methods. The values ± standard deviation are reported as the average of at least three independent determinations.
Fig. 5. Specificity of monoclonal antibodies raised against the Sf21 cathepsin. The cathepsin zymogen and mature enzyme forms were analyzed by immunoblotting using the monoclonal antibodies MAb 1 and MAb 2. Lanes 1 and 5 contain 20 ng zymogen, lanes 2 and 6 contain 100 ng zymogen. Lanes 3 and 7 contain 100 ng of fully activated, mature enzyme. Partially activated cathepsin (400 ng) was run in lanes 4 and 8. The positions of molecular mass standards (kDa) are shown on the left. The brackets at the bottom indicate that lanes 1–4 were incubated with MAb 1 primary antibody, and lanes 5–8 were incubated with MAb 2 primary antibody.
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not detected by MAb 2. Because MAb 1, but not MAb 2, can detect activation intermediates as large as 40 kDa, it is likely that the MAb 2 epitope is in the NH2-terminal portion of the cathepsin propeptide. MAb 1 and MAb 2 were also used for immunoblot analysis of conditioned media samples from the insect cell lines most commonly used in expression systems. The highest expression level was observed for Sf21 cells, and the procathepsin L was also easily detected in Sf9 conditioned medium. Trichoplusia ni cells secrete a 52-kDa protein that is recognized by both MAb 1 and MAb 2, but it is secreted at a lower level than the Sf9 and Sf21 procathepsin L (data not shown). Therefore, it is apparent that the cells currently used in most insect expression systems secrete procathepsin L. In summary, we have purified and characterized a secreted cathepsin L homologue that is produced by Sf21 insect cells. The insect protease closely resembles mammalian cathepsin L in amino acid sequence, substrate specificity, and inhibitor profile. It differs from its mammalian counterparts in being oligomeric rather than a monomer. The presence of this enzyme poses problems for those using insect cell systems for the heterologous expression of recombinant proteins. The enzyme is a potent protease, readily degrading recombinant proteins expressed in insect cell systems, particularly at acidic pH. The Sf21 cathepsin L tends to bind immobilized metal affinity resins charged with Ni2+ ions, complicating the purification of histidine-tagged proteins. In this study, we have described how to identify the insect cathepsin by its enzymatic activity and by its immunoreactivity, and identified a number of potent inhibitors of the enzyme. The information presented here can be used by those employing insect cell expression systems to detect the presence of the insect cathepsin L and to minimize its proteolytic activity during protein expression experiments. Acknowledgments We wish to thank the numerous individuals from various departments at R&D Systems, who have contributed to this work in the course of product development. References [1] H.A. Chapman, R.J. Riese, G.-P. Shi, Annu. Rev. Physiol. 59 (1997) 63–88. [2] V. Turk, B. Turk, D. Turk, EMBO J. 20 (2001) 4629–4633. [3] G.-P. Shi, J.A. Villadangos, G. Dranoff, C. Small, L. Gu, K.J. Haley, R. Riese, H.L. Ploegh, H.A. Chapman, Immunity 10 (1999) 197–206. [4] G.-P. Shi, R.A. Bryant, R. Riese, S. Verhelst, C. Driessen, Z. Li, D. Bro¨mme, H.L. Ploegh, H.A. Chapman, J. Exp. Med. 191 (2000) 1177–1186. [5] C.T. Pham, T.J. Ley, Proc. Natl. Acad. Sci. USA 96 (1999) 8627– 8632. [6] C. Tepel, D. Bro¨mme, V. Herzog, C. Brix, J. Cell Sci. 113 (2000) 4487–4498.
[7] G. Guncˇar, M. Podobnik, J. Pungercˇar, B. Sˇtrukelj, V. Turk, D. Turk, Structure 6 (1998) 51–61. [8] P. Saftig, E. Hunzicker, O. Wehmeyer, S. Jones, A. Boyde, W. Rommerskirch, J.D. Moritz, P. Schu, K. von Figura, Proc. Natl. Acad. Sci. USA 95 (1998) 13453–13458. [9] K.J. Homma, in: A.J. Barrett, N.D. Rawlings, J.F. Woessner (Eds.), Handbook of Proteolytic Enzymes, second ed., Elsevier Academic Press, London, 2004, pp. 1189–1192. [10] I. Matsumoto, Y. Emori, K. Abe, S. Arai, J. Biochem. 121 (1997) 464–476. [11] T. Kageyama, S.Y. Takahashi, Eur. J. Biochem. 193 (1990) 203– 210. [12] Y. Yamamoto, K. Takimoto, S. Izumi, M. Toriyama-Sakurai, T. Kageyama, S.Y. Takahashi, J. Biochem. 116 (1994) 1330–1335. [13] K.-I. Homma, S. Kurata, S. Natori, J. Biol. Chem. 269 (1994) 15258– 15264. [14] I. Fujii-Taira, Y. Tanaka, K.J. Homma, S. Natori, J. Biochem. 128 (2000) 539–542. [15] S. Natori, H. Shiraishi, S. Hori, A. Kobayashi, Dev. Comp. Immunol. 23 (1999) 317–328. [16] I. Vaughn, R.H. Goodwin, G.L. Tompkins, P. McCawley, In Vitro 13 (1977) 213–217. [17] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Nucleic Acids Res. 25 (1997) 3389–3402. [18] J.D. Thompson, D.G. Higgins, T.J. Gibson, Nucleic Acids Res. 22 (1994) 4673–4680. [19] R.L. Wolz, J.S. Bond, Methods Enzymol. 248 (1995) 325–345. [20] C.G. Knight, Methods Enzymol. 248 (1995) 85–101. [21] A. Hernandez, M.T. Ruiz, Bioinformatics 14 (1998) 227–228. [22] R. Menard, E. Carmona, S. Takebe, E. Dufour, C. Plouffe, P. Mason, J.S. Mort, J. Biol. Chem. 273 (1998) 4478–4484. [23] T. Vernet, H.E. Khouri, P. Laflamme, D.C. Tessier, R. Musil, B.J. Gour-Salin, A.C. Storer, D.Y. Thomas, J. Biol. Chem. 266 (1991) 21451–21457. [24] D. Bro¨mme, P.R. Bonneau, P. Lachance, B. Wiederanders, H. Kirschke, C. Peters, D.Y. Thomas, A.C. Storer, T. Vernet, J. Biol. Chem. 268 (1993) 4832–4838. [25] D. Bro¨mme, P.R. Bonneau, P. Lachance, A.C. Storer, J. Biol. Chem. 269 (1994) 30238–30242. [26] D. Bro¨mme, K. Okamoto, B.B. Wang, S. Biroc, J. Biol. Chem. 271 (1996) 2126–2132. [27] D.K. Na¨gler, W. Tam, A.C. Storer, J.C. Krupa, J.S. Mort, R. Menard, Biochemistry 38 (1999) 4868–4874. [28] H.E. Khouri, C. Plouffe, S. Hasnain, T. Hirama, A.C. Storer, R. Menard, Biochem. J. 275 (1991) 751–757. [29] H. Kirschke, in: A.J. Barrett, N.D. Rawlings, J.F. Woessner (Eds.), Handbook of Proteolytic Enzymes, second ed., Elsevier Academic Press, London, 2004, pp. 1097–1102. [30] I. Schechter, A. Berger, Biochem. Biophys. Res. Commun. 27 (1967) 157–162. [31] D. Bro¨mme, A. Steinert, S. Friebe, S. Fittkau, B. Wiederanders, H. Kirschke, Biochem. J. 264 (1989) 475–481. [32] A.J. Barrett, H. Kirschke, Methods Enzymol. 80 (1981) 535–561. [33] A.J. Barrett, M.E. Davies, A. Grubb, Biochem. Biophys. Res. Commun. 120 (1984) 631–636. [34] J.P. Freije, M. Balbı´n, M. Abrahamson, G. Velasco, H. Dalbøge, A. Grubb, C. Lo´pez-Otı´n, J. Biol. Chem. 268 (1993) 15737–15744. [35] M. Balbı´n, A. Hall, A. Grubb, R.W. Mason, C. Lo´pez-Otı´n, M. Abrahamson, J. Biol. Chem. 269 (1994) 23156–23162. [36] G.D. Green, A.A. Kembhavi, M.E. Davies, A.J. Barrett, Biochem. J. 218 (1984) 939–946. [37] D. Bro¨mme, R. Rinne, H. Kirschke, Biomed. Biochim. Acta 50 (1991) 631–635. [38] P. Lindahl, M. Abrahamson, I. Bjo¨rk, Biochem. J. 281 (1992) 49–55. [39] M. Abrahamson, M. Alvarez-Fernandez, C.-M. Nathanson, Biochem. Soc. Symp. 70 (2003) 179–199.
Characterization of cathepsin L secreted by Sf21 insect cells Gary D. Johnson *, Weiping Jiang R&D Systems, Inc. 614 McKinley Pl. NE, Minneapolis, MN 55413, USA Received 26 July 2005, and in revised form 20 September 2005 Available online 21 October 2005
Abstract Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni2+ affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed. 2005 Elsevier Inc. All rights reserved. Keywords: Cysteine proteases; Cathepsin L; Subsite specificity; Cystatins; Monoclonal antibodies
The majority of known cathepsins are peptidases that utilize a catalytic cysteine residue in their active site. These enzymes, structurally related to papain, belong to the C1 peptidase family [1]. While C1 cathepsins are generally endopeptidases, there are known examples of exopeptidases in the family (e.g., cathepsins C and X) [2]. Most cathepsins are present in lysosomes, where they play a major role in intracellular protein degradation [1,2]. Cathepsins are also present in other cellular compartments, or are secreted and associated with the plasma membrane, allowing them to perform additional functions. Cathepsins are known to be involved in antigen presentation [1,3,4], zymogen activation [5], and prohormone processing [6]. Most cathepsins are ubiquitously expressed, but others have a more restricted tissue distribution [1,2], resulting in specialized functions. For example, cathepsin K is secreted by osteoblasts and plays a major role in bone resorption. Cathepsin K deficiencies are responsible for the bone diseases pycnodysostosis and osteopetrosis [7,8].
*
Corresponding author. E-mail address: [email protected] (G.D. Johnson).
0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.09.011
Cathepsins of the C1 family are expressed by most multicellular eukaryotes, including insects. The majority of insect cathepsins are homologous to mammalian cathepsins B and L [9]. A number of secreted insect cathepsins may be involved in the protein degradation that occurs during food digestion [10], embryogenesis [11,12], and metamorphosis [13,14]. Insect cathepsins may also be a part of a defense system, eliminating foreign proteins by degradation [15]. Sf21 insect cells are derived from pupal ovarian cells of Spodoptera frugiperda (Fall Armyworm) [16]. These cells are frequently used for the heterologous expression of recombinant proteins. During several attempts at the expression of mammalian cathepsins in the Sf21 cell line, we observed that the cell line secreted a protease with properties similar to mammalian cathepsins that also co-purified with the human enzymes. The insect protease was purified and biochemically characterized. The pH/activity and inhibitor profiles showed that the insect protease is a cysteine protease of the C1 family. Substrate specificity studies and amino acid sequence data revealed that the protease is an insect homologue of mammalian cathepsin L. Monoclonal antibodies were raised against the pro and
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mature forms of the insect cathepsin L. These antibodies are useful for the detection of the enzyme, and possibly its purification by immunoaffinity methods. Materials and methods Protein purification All purification steps were performed at 6–10 C. Sf21 cells were grown in Ex-cell 401 serum-free insect medium from JRH Biosciences (Lenexa, KS) to a density of 1.4 · 107 cells/ml. Conditioned medium was harvested, concentrated 10-fold by tangential flow ultrafiltration, and the pH was adjusted to 7.5 with 1 N NaOH. Insoluble material was removed by centrifugation at 21,000g. The soluble protein was diluted 4-fold with cold 5 mM Hepes,1 0.1 M NaCl, pH 7.5, then loaded onto an SP Sepharose (GE Healthcare, Piscataway, NJ) column equilibrated in 25 mM Hepes, 0.1 M NaCl, pH 7.5 (buffer A). Bound proteins were eluted using a linear gradient of NaCl (0.1–1 M) in buffer A, with the fractions collected in tubes containing 1 M NaCl to minimize precipitation of the eluted protein. Fractions were assayed, after autoactivation, using the substrate benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide (Z-FR-MCA) at 10 lM in 50 mM NaOAc, 1 mM DTT, pH 5.0. The fractions with high specific activity were pooled. A 5-ml chelating Sepharose column (GE Healthcare) was charged with Ni2+ ions, then equilibrated in 25 mM Hepes, 0.5 M NaCl, and 10 mM imidazole, pH 7.5 (buffer B). Protein pooled from the SP Sepharose column was diluted 4-fold in buffer B and loaded onto the chelating Sepharose column. The column was washed with buffer B before the elution of bound proteins with 0.2 M imidazole, 0.5 M NaCl, pH 7.5. The protein eluted from the chelating Sepharose column was concentrated to 4 ml by ultrafiltration using a 30-kDa NMWL Ultracel membrane in an Amicon stirred cell from Millipore (Billerica, MA), and insoluble material removed by centrifugation at 16,000g. The concentrated soluble protein was then subjected to gel permeation chromatography on a 2.5 · 90 cm Superdex 200 (GE Healthcare) column equilibrated in 25 mM Hepes, 0.5 M NaCl, and 0.1% Brij 35, pH 7.5. The Superdex 200 column was calibrated with molecular mass standards (ferritin, catalase, aldolase, albumin, ovalbumin, and chymotrypsinogen A) purchased from GE Healthcare to determine the molecular mass of the native enzyme. SDS–PAGE was performed using Tris–glycine gels with molecular mass standards purchased from BioRad Laboratories (Hercules, CA).
1
Abbreviations used: Boc, tert-butoxycarbonyl; DTT, dithiothreitol; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; Hepes, N-(2-hydroxyethyl)piperazine-N 0 -(2-ethanesulfonic acid); MCA, 4-methyl-7-coumarylamide; LC–MS/MS, liquid chromatography nano-spray mass spectrometry/mass spectrometry; Mes, 2-(N-morpholino)ethanesulfonic acid; NaOAc, sodium acetate; RFU, relative fluorescence unit; Z, benzyloxycarbonyl.
Amino acid sequence analysis The purified enzyme in solution was submitted to the Molecular Structure Facility of the University of California at Davis for NH2-terminal sequence analysis by the Edman degradation procedure. The resulting amino acid sequence was used to search protein databanks for sequence matches using the Blastp program [17]. The selected sequence matches were aligned using Clustal W [18]. The purified protein was also submitted to the Molecular Biology and Biophysics Proteomic Analysis Core facility at the University of Minnesota, St. Paul for internal peptide analysis involving in-gel trypsin digestion followed by liquid chromatography nano-spray mass spectrometry/ mass spectrometry (LC–MS/MS). Determination of carbohydrate content The carbohydrate content of the purified Sf21 protease was measured using a glycoprotein carbohydrate estimation kit purchased from Pierce Biotechnology (Rockford, IL). The Sf21 protease and standard proteins of known carbohydrate content were first oxidized by sodium metaperiodate, then the resulting aldehydes were reacted with an aldehyde detection reagent as recommended by the manufacturer. The absorbance at 550 nm was then measured for the standard proteins and the Sf21 protease, and a standard curve was generated to relate the absorbance to percentage carbohydrate content. Lysozyme was used as a negative control. Positive controls were ovalbumin, apotransferrin, fetuin, and a1-acid glycoprotein. Enzyme autoactivation and assay optimization To determine the optimal conditions for autoactivation of the cathepsin, purified cathepsin was diluted >20-fold (to 0.02 mg/ml) in 0.1 M NaOAc, and 10 mM DTT, pH 5.0, then incubated at 37 C to permit autoactivation. At various time points aliquots of the incubation mixture were removed for immediate assay using the Z-FR-MCA substrate. At each time point, 2 lg aliquots of protein were removed and the protein was precipitated by the addition of trichloroacetic acid to a final concentration of 10%. The precipitated protein was saved for analysis by SDS– PAGE under reducing conditions. The activity of the Sf21 protease was examined under conditions in which the pH and ionic strength of the reaction buffer were varied. Purified enzyme autoactivated in 0.1 M NaOAc, 10 mM DTT, pH 5.0, was diluted 20-fold in 10 mM DTT, then diluted an additional 10-fold into microplate wells containing NaOAc buffers (pH 3–5) or Mes buffers (pH 5.5–7). Buffer concentrations were 55 mM, and the final DTT concentration was 1 mM. NaCl concentration was varied at each pH from 0–1 M. Reactions were initiated by the addition of Z-FR-MCA substrate to 10 lM final concentration. Reaction rates were monitored by measuring the rates of liberation of free 7-
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amino-4-methylcoumarin on a SpectraMax Gemini fluorescence plate reader from Molecular Devices (Sunnyvale, CA) using the excitation and emission wavelengths of 380 and 460 nm, respectively. Substrate specificity of the Sf21 cathepsin Z-LR-MCA and Z-FR-MCA substrates were obtained from R&D Systems (Minneapolis, MN). All other peptidyl-MCA substrates were purchased from Bachem (Torrance, CA). Peptidyl-MCA substrates were used at 10 lM in a buffer consisting of 50 mM NaOAc, 1 mM DTT, pH 5.0. Reactions were started by the addition of activated enzyme to microplate wells, and the resulting fluorescence increases were recorded using a fluorescence plate reader at 24 C. Relative fluorescence units (RFU) were converted to moles of product using a conversion factor generated using purified 7-amino-4-methylcoumarin from Sigma (St. Louis, MO) as the standard. Azocasein (Sigma) degradation assays were performed as described by Wolz and Bond [19], except that 50 mM Mes, 2.5 mM DTT, pH 5.0 was used as the assay buffer.
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cells. Conditioned media samples from the resulting hybridomas were screened for binding to the Sf21 cathepsin by Western blot analysis. Sf21 conditioned medium was run on 12% Tris–glycine gels, the fractionated proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories), and incubated with hybridoma culture medium diluted in Tris-buffered saline containing 1% nonfat dried milk and 0.1% Tween 20. The immunoblots were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (R&D Systems) and developed using Western Glo chemiluminescent detection reagents (R&D Systems). Hybridoma culture media samples that reacted with the 48-kDa Sf21 protease band, but not with other Sf21 secreted proteins, were judged to be positive. Positive hybridoma clones were expanded and the secreted antibodies were purified by Protein G affinity chromatography. Immunoblot analysis of the purified monoclonal antibodies was used to identify antibodies specific for the pro and mature domains of the Sf21 cathepsin. Results and discussion Enzyme purification
Determination of kinetic constants KM and Vmax values for cathepsin hydrolysis of Z-FRMCA were determined using a concentration of 0.484 nM of enzyme in a buffer composed of 50 mM NaOAc, 1 mM DTT, pH 5.0, at 24 C. Substrate concentrations were varied from 2 to 100 lM. Initial rate data were recorded as RFU s1 using a fluorescence plate reader. Conversion factors for RFU to moles of product were determined for each substrate concentration to correct for inner filter effects. The corrected initial rate data were fit to the Michaelis–Menten equation by nonlinear regression analysis using Kaleidagraph software from Synergy Software (Reading, PA). The value of kcat was calculated from Vmax using the enzyme concentration determined by active site titration with the inhibitor E-64 as described by Knight [20]. Recombinant human cystatins (A, B, C, D, E/M, F, S, SA, and SN) were obtained from R&D Systems. Ki values for the cystatins were determined using a constant concentration of 0.97 nM of activated cathepsin, and cystatins were varied 20–60-fold in concentration. Enzyme and inhibitor were preincubated for 30 min at 24 C before the addition of Z-FR-MCA substrate. The initial reaction rates were measured at three substrate concentrations (5, 10, and 25 lM) for each inhibitor concentration using a fluorescence plate reader. The resulting data were fit to a model for competitive inhibition (v = Vmax)/(1 + KM/ [S] + (1 + [I]/Ki)) using a computer program developed by Hernandez and Ruiz [21]. Monoclonal antibody production and characterization B cells obtained from mice immunized with purified Sf21 secreted cathepsin were fused with mouse myeloma
The Sf21 protease was purified to electrophoretic homogeneity using ion-exchange chromatography (SP Sepharose), immobilized metal affinity chromatography (chelating Sepharose), and gel-permeation chromatography (Superdex 200). All purification steps were carried out at pH 7.5 to prevent the autoactivation and subsequent degradation of the enzyme that occurs at acidic pH. SDS– PAGE analysis of the purification pools under reducing conditions is shown in Fig. 1. The purified protease has
Fig. 1. SDS–PAGE analysis of Sf21 protease purification. Lane 1, conditioned medium; lane 2, SP Sepharose pool; lane 3, chelating Sepharose pool; and lane 4, Superdex 200 pool. Lane M contains molecular mass standards. Molecular masses of the standards (kDa) are displayed on the right. Proteins were visualized by silver staining.
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Table 1 Purification of Sf21 protease from 10 L of conditioned medium Pool
Total protein (mg)
Total activity (lmol min1)
Specific activity (lmol mg1 min1)
Purification factor
Yield (%)
Conditioned medium SP Sepharose Chelating Sepharose Superdex 200
1620 54 31 15.8
510 160 200 160
0.32 3.0 6.6 10.1
1 9.6 20.8 32
100 31 39 31
an apparent subunit molecular mass of 48 kDa. From lane 1, it is evident that the enzyme is one of the more abundant proteins secreted by the Sf21 cells, as the 48 kDa protein band is clearly visible in the mixture of proteins in the conditioned medium. The protease is substantially purified by the SP Sepharose step (lane 2), with the remaining contaminants removed by the chelating Sepharose and Superdex 200 steps (lanes 3 and 4). The results of a typical purification are summarized in Table 1. Activity measurements of several batches of the conditioned medium using the Z-FR-MCA substrate indicate that the enzyme is expressed at a level of 5 mg/L or greater. At least half of this activity was generally lost during the SP Sepharose step due to the tendency of the enzyme to precipitate under conditions of low ionic strength at neutral pH. After the SP Sepharose step, the inclusion of 0.5 M NaCl in all buffers minimized precipitation of the enzyme. Typically, a yield of 1.5–2.0 mg of purified enzyme per liter of conditioned medium was achieved. The activity of the purified protease was completely inhibited by E-64, but no inhibition was observed using either the serine protease inhibitor 3,4-dichloroisocoumarin or the aspartic protease inhibitor Pepstatin A (data not shown), evidence that the enzyme is a member of the C1 cysteine peptidase family. The subunit molecular mass of the Sf21 protease was estimated to be 48 kDa by SDS–PAGE, but the enzyme behaved as a much larger protein during the gel permeation chromatography step. The Superdex 200 column used for purification of the enzyme was calibrated with protein molecular mass standards. From these data, a molecular mass of 405 kDa was calculated for the native Sf21 secreted protease. Therefore, the protease is oligomeric, having a mass expected for an octamer. Amino acid sequence analysis of the purified Sf21 secreted protease
the cotton bollworm cathepsin L peptides with corresponding masses, and that the two proteins share a very high degree of amino acid sequence identity. Fig. 2 displays an alignment of the four Sf21 protease peptides with the amino acid sequences of procathepsin L from cotton bollworm, fruit fly, and human. The NH2-terminal sequence of the Sf21 protease, located at the beginning of the propeptide, is very similar to the insect sequences, but has little identity with the human sequence. However, the tryptic peptides, all located within the catalytic domain, display a much greater degree of interspecies identity. These peptides are 100, 68, and 76% identical to the corresponding fruit fly sequences, and 58, 53, and 59% identical to the corresponding human procathepsin L sequences. This high degree of sequence identity is consistent with the hypothesis that the Sf21 secreted protease is an insect cathepsin L. Assuming 100% sequence identity with the cotton bollworm protein, the Sf21 protein is initially synthesized as a 341 amino acid preproenzyme, becoming a 325 residue zymogen after removal of a 16 residue signal sequence. By analogy with human cathepsin L, generation of the mature, active Sf21 cathepsin L would require the removal of a 110–115 residue propeptide [22]. Determination of carbohydrate content The predicted molecular mass of the Sf21 cathepsin zymogen is approximately 35 kDa. However, the subunit molecular mass was estimated to be 48 kDa by SDS– PAGE, indicating that the protein may be glycosylated. The presence of carbohydrate modifications on the protease was confirmed using periodate oxidation of the purified cathepsin, followed by detection of the resulting aldehydes. From a comparison with standard proteins of known carbohydrate content, it was estimated that the Sf21 cathepsin zymogen contains >25% carbohydrate. Autoactivation of the purified Sf21 cathepsin L
The NH2-terminal sequence of the Sf21 secreted protease zymogen was determined. The NH2-terminal sequence (VSLLDLVRE) is identical to the NH2-terminal sequence of a putative procathepsin L from the cotton bollworm as deduced from its cDNA (GenBank Accession No. AAQ75437). The molecular masses of three tryptic peptides derived from the Sf21 protease, a total of 48 amino acid residues, were determined to be the same as three deduced tryptic peptides of the cotton bollworm cathepsin L. Therefore, it is probable that these three tryptic peptides of the Sf21 protease are identical in amino acid sequence to
Preliminary tests revealed that the Sf21 cathepsin L remains stable as the zymogen at pH 7.5, but undergoes autoactivation at pH 5 or lower, and that complete activation required the presence of a reducing agent such as DTT. SDS–PAGE analysis of the time course for activation under the conditions described is shown in Fig. 3. Aliquots at each time point were also monitored for activity using the substrate Z-FR-MCA (data not shown). The purified enzyme is initially present as the 48-kDa proform, which has only 2% of the activity of the fully activated
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Fig. 2. Amino acid sequence alignments of procathepsin L from Sf21, cotton bollworm (cb, AAQ75437), fruit fly (ff, Q95029), and human (h, P07711). Identical and similar residues are indicated by * and :, respectively. The sequences underlined in cb are perfect matches to the sequences determined for the Sf21 protease. The NH2-terminal sequence of the Sf21 proenzyme was determined from the intact protein in solution. The masses (Mr experimental/ calculated) listed above the three internal peptides were the results from LC–MS/MS analysis of tryptic peptides. The Cys residue in the last peptide was modified by a carbamidomethyl group.
Fig. 3. Autoactivation of the Sf21 cathepsin at pH 5.0. The numbers above each lane indicate the time point in minutes at which aliquots were removed from the autoactivation reaction for SDS–PAGE analysis. Molecular mass standards were run in lane M, with molecular masses of the standards (kDa) shown on the left.
enzyme. Activation proceeded slowly for the first 20 min, until the proform was partially converted to a 34-kDa species. Within 30 min, much of the proform was converted to the 34-kDa form, and a 32-kDa form was also beginning to appear. Processing was complete by 40 min of incubation, with the conversion of the proform to the 32 kDa mature
enzyme form. From activity measurements it was apparent that both the 34- and 32-kDa enzyme forms are highly active (data not shown). Trace amounts of propeptidesized fragments (14 and 16 kDa) were visible at the 30and 40-min time points on overexposed gels, but were quickly degraded and no longer detected with silver staining by the 60-min time point. It is clear that the Sf21 protease proform efficiently transforms itself into the mature, active enzyme form under acidic conditions. Autocatalytic processing of the precursor to the mature enzyme form by removal of the NH2-terminal propeptide has also been reported for the cysteine endopeptidases papain [23] and cathepsin L [22] under similar conditions. By contrast, procathepsins S and B are able undergo partial activation to intermediate forms [23,24] which can be trimmed by exopeptidases to generate the mature enzyme form. Dependence of Sf21 cathepsin L activity on pH and ionic strength The common cathepsin substrate Z-FR-MCA was used to examine the effects of pH and ionic strength on the activity of the Sf21 cathepsin L. The resulting data are presented in the three-dimensional bar graph of Fig. 4, with
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G.D. Johnson, W. Jiang / Archives of Biochemistry and Biophysics 444 (2005) 7–14 Table 2 Rates of hydrolysis of peptidyl-MCA substrates by the secreted Sf21 cathepsin
Fig. 4. Effects of pH and NaCl concentration on Sf21 cathepsin hydrolysis of Z-FR-MCA. NaCl concentrations (M) are shown on the left axis and coded in the bar graph: black, 0 M; white, 0.1 M; diagonal stripes, 0.25 M; and stippled, 1.0 M. Buffer pH is shown on the center axis. Enzyme activity in relative fluorescence U/s (RFU/s) is displayed on the right axis.
activity displayed as relative fluorescence U/s (RFU/s). The enzyme is active in the pH range 3.0–6.5, with optimal activity at pH 5.0–6.0. pH/activity profiles have been determined for a number of cathepsins using the Z-FR-MCA substrate. Most of these enzymes were observed to be active in the pH range 3.5–8.5. These enzymes include cathepsins S [25], K [26,27], and B [28]. Cathepsin L, however, is known to be active in the pH range 3.0–6.5 [29]. The Sf21 cathepsin most resembles cathepsin L in its pH/ activity profile. Activity was greatest at low ionic strength, decreasing by about 40% at most pH values tested as NaCl was added to 0.1 and 0.25 M in the reactions. Raising (NaCl] from 0.25 to 1.0 M resulted in slight activity increases in the pH 4.0–5.0 range, but resulted in decreased activity at pH 3.0 and at pH 5.0 or higher. As a whole, the effect of ionic strength on the Sf21 cathepsin activity is modest near the optimal pH, but significant at low pH and near neutral pH. Subsite specificity mapping using peptidyl-MCA substrates Peptidyl-MCA substrates are commonly used to assay the activity of cysteine proteases of the C1 family. A variety of peptidyl-MCA substrates, all having a common P01 group (MCA), were used to probe the P1 and P2 subsite specificity of the Sf21 cathepsin (see [30] for a discussion of peptidase subsite notation). One advantage of using these substrates is the requirement that the 7-amino-4methylcoumarin (P01 ) group must be released in order for a fluorescence increase to occur, thereby defining the positions of P1 and P01 in the substrates. The peptidyl-MCA substrates were reacted with the insect cathepsin, and the rates are expressed as specific activities (lmol mg1 min1) in Table 2. The substrates used are ranked from highest to lowest specific activity in the table. The enzyme exhibits a clear preference for Arg residues in the P1 position, demon-
Substrate
Specific activity (lmol mg1 min1)
Z-FR-MCA Z-LR-MCA PFR-MCA succinyl-AFK-MCA LY-MCA Boc-RR-MCA Succinyl-LLVY-MCA Boc-RVRR-MCA Boc-VPR-MCA R-MCA GR-MCA Z-GGR-MCA Boc-IEGR-MCA Z-GP-MCA Succinyl-AAPF-MCA Z-AAN-MCA Acetyl-IEPD-MCA
11.0 8.7 3.9 0.38 0.36 0.03 0.012 0.010 0.008 ND ND ND ND ND ND ND ND
Conditions for hydrolysis are described in Materials and methods. The specific activity values reported are the average of three separate determinations. ND, no hydrolysis detected.
strated by the high rates of hydrolysis of Z-FR-MCA, ZLR-MCA, and PFR-MCA. Lys and the hydrophobic residue Tyr were also accepted at P1 (succinyl-AFK-MCA and LY-MCA), but these substrates were hydrolyzed at a rate at least an order of magnitude more slowly than the best substrates having Arg at P1. No hydrolysis was detected for the substrates having Pro, Asn, or Asp in the P1 position. Despite the preference for P1 Arg residues, no hydrolysis of Arg-MCA was detected, indicating that the enzyme is not an effective aminopeptidase. A comparison of the substrates with P1 Arg residues indicates a preference for large hydrophobic residues in the P2 position, as shown by the presence of Phe or Leu at P2 for all five of the substrates that were hydrolyzed at high rates. Furthermore, comparison of the hydrolysis rates of Z-FR-MCA and ZLR-MCA reveals a preference for Phe at P2. This pattern of subsite specificity is very similar to that of the mammalian cysteine proteases cathepsin B and cathepsin L [25,31]. These two cathepsins, however, differ in their hydrolysis of substrates with P2 Arg residues. Cathepsin B hydrolyzes P2 Arg substrates with rates comparable to P2 Phe and Leu substrates [32], but P2 Arg substrates are hydrolyzed relatively slowly by cathepsin L [26,32]. In the present survey of peptidyl-MCA substrates for the Sf21 secreted cysteine protease, it is clear that peptides with a P2 Arg residue (Boc-RR-MCA and Boc-RVRR-MCA) are cleaved at much lower rates than substrates with hydrophobic P2 residues. Therefore, the results of this study indicate that the Sf21 cathepsin is an endopeptidase with subsite specificity closely resembling that of cathepsin L. The values of KM and kcat were determined for Z-FRMCA, the best of the peptidyl-MCA substrates tested. The KM was determined to be 11 lM (average of five experiments, data not shown), a value typical for cathepsins of the C1 cysteine peptidase family, which generally fall in
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the 5–20 lM range [24–26]. The kcat/KM of 1.6 · 106 M1 s1 is intermediate to those reported for human cathepsins B and L [25,26]. Protease activity of the purified Sf21 cathepsin The hydrolysis of the protein substrate azocasein by the purified cathepsin was measured as described in Materials and methods. It was determined that 1 mg of the cathepsin is able to solubilize 23.7 mg azocasein min1 at 37 C. Therefore, the Sf21 cathepsin L is a very active protease as well as a peptidase. Cystatin inhibitors of the Sf21 cathepsin A panel of cystatins was tested for the inhibition of the Sf21 cathepsin. Initial tests revealed that human cystatins A, B, C, D, E/M, F, SA, and SN are all potent inhibitors of the enzyme, but that human cystatin S inhibits it only weakly. The equilibrium constant for dissociation of complexes (Ki) between the Sf21 cathepsin and various cystatins was determined for the more potent inhibitors, and the results are presented in Table 3. Although cystatin C appeared to have a Ki value in the range of 0.1–0.5 nM, a Ki for this inhibitor is not included in Table 3 because the data obtained were not a precise fit to the model for competitive inhibition. Of the cystatins for which accurate Ki values could be determined, cystatins A, B, E/M, F, SA, and SN were found to be subnanomolar inhibitors of the Sf21 cathepsin, with Ki values in the range of 0.19– 0.51 nM. These cystatins are as potent as E-64, the active site-directed inhibitor that is frequently used for the inhibition of papain-like cysteine proteases. We have determined a Ki value of 0.59 ± 0.13 nM for E-64 inhibition of the Sf21 cathepsin L (data not shown). Cystatin D, with a Ki of 6.5 nM, was somewhat less potent than the other cystatins included in Table 3. The Ki values for the inhibition of mammalian cathepsins B, H, L, and S by cystatins A, B, C, and D have been previously determined [33–38] and summarized by Abrahamson et al. [39]. A comparison of these data with the present study indicates that the Sf21 cathepsin exhibits an inhibitor profile closely resembling that of cathepsin H, for which Ki values of 0.31, 0.58, 0.28, and 7.5 nM have been determined for cystatins A,
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B, C, and D, respectively. The cystatin inhibition profile of the Sf21 cathepsin is also similar to those of cathepsins L and S, except for a less potent inhibition of the insect protease by cystatin C. The cystatin inhibitor profile of the Sf21 cathepsin is very distinct from that of cathepsin B. Specificity of monoclonal antibodies raised against the insect cathepsin The majority of the monoclonal antibodies raised against the insect cathepsin were found to recognize both the zymogen and the mature, activated enzyme as judged by Western blot analysis. However, one clone produced an antibody specific for the cathepsin propeptide. A comparison of the specificity of two of the monoclonal antibodies as assayed by immunoblotting is shown in Fig. 5. Primary antibodies MAb 1 (clone 193702) and MAb 2 (clone 193720) at 1 lg/ml were used to detect the Sf21 cathepsin zymogen and autoactivated mature enzyme. The purified zymogen was loaded in lanes 1 and 5 at 20 ng, and at 100 ng in lanes 2 and 6. Fully activated, mature cathepsin was loaded at 100 ng in lanes 3 and 7. Partially activated cathepsin was loaded at 400 ng in lanes 4 and 8. MAb 1 was used as the primary antibody for lanes 1-4, MAb 2 was the primary antibody for lanes 5–8. Both antibodies are able to detect as little as 20 ng of the 48-kDa zymogen, but MAb 1 is slightly more sensitive, as its signal in lane 1 is stronger than lane 5. Only MAb 1 is able to detect the 32 kDa mature enzyme, judging from the strong signal in lane 3 compared to the absence of a signal in lane 7. It is apparent that the epitope for MAb 2 lies partly or entirely in the cathepsin propeptide because of its failure to recognize the mature form of the enzyme. Comparing lanes 4 and 8, it is evident that MAb 1 recognizes several activation intermediates between 34 and 40 kDa that are
Table 3 Inhibition of the Sf21 secreted cathepsin by human cystatins Cystatin
Ki (nM) ± standard deviation
A B D E/M F SA SN
0.21 ± 0.026 0.46 ± 0.040 6.5 ± 1.5 0.19 ± 0.054 0.21 ± 0.025 0.51 ± 0.11 0.27 ± 0.021
Ki values were determined as described in Materials and methods. The values ± standard deviation are reported as the average of at least three independent determinations.
Fig. 5. Specificity of monoclonal antibodies raised against the Sf21 cathepsin. The cathepsin zymogen and mature enzyme forms were analyzed by immunoblotting using the monoclonal antibodies MAb 1 and MAb 2. Lanes 1 and 5 contain 20 ng zymogen, lanes 2 and 6 contain 100 ng zymogen. Lanes 3 and 7 contain 100 ng of fully activated, mature enzyme. Partially activated cathepsin (400 ng) was run in lanes 4 and 8. The positions of molecular mass standards (kDa) are shown on the left. The brackets at the bottom indicate that lanes 1–4 were incubated with MAb 1 primary antibody, and lanes 5–8 were incubated with MAb 2 primary antibody.
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not detected by MAb 2. Because MAb 1, but not MAb 2, can detect activation intermediates as large as 40 kDa, it is likely that the MAb 2 epitope is in the NH2-terminal portion of the cathepsin propeptide. MAb 1 and MAb 2 were also used for immunoblot analysis of conditioned media samples from the insect cell lines most commonly used in expression systems. The highest expression level was observed for Sf21 cells, and the procathepsin L was also easily detected in Sf9 conditioned medium. Trichoplusia ni cells secrete a 52-kDa protein that is recognized by both MAb 1 and MAb 2, but it is secreted at a lower level than the Sf9 and Sf21 procathepsin L (data not shown). Therefore, it is apparent that the cells currently used in most insect expression systems secrete procathepsin L. In summary, we have purified and characterized a secreted cathepsin L homologue that is produced by Sf21 insect cells. The insect protease closely resembles mammalian cathepsin L in amino acid sequence, substrate specificity, and inhibitor profile. It differs from its mammalian counterparts in being oligomeric rather than a monomer. The presence of this enzyme poses problems for those using insect cell systems for the heterologous expression of recombinant proteins. The enzyme is a potent protease, readily degrading recombinant proteins expressed in insect cell systems, particularly at acidic pH. The Sf21 cathepsin L tends to bind immobilized metal affinity resins charged with Ni2+ ions, complicating the purification of histidine-tagged proteins. In this study, we have described how to identify the insect cathepsin by its enzymatic activity and by its immunoreactivity, and identified a number of potent inhibitors of the enzyme. The information presented here can be used by those employing insect cell expression systems to detect the presence of the insect cathepsin L and to minimize its proteolytic activity during protein expression experiments. Acknowledgments We wish to thank the numerous individuals from various departments at R&D Systems, who have contributed to this work in the course of product development. References [1] H.A. Chapman, R.J. Riese, G.-P. Shi, Annu. Rev. Physiol. 59 (1997) 63–88. [2] V. Turk, B. Turk, D. Turk, EMBO J. 20 (2001) 4629–4633. [3] G.-P. Shi, J.A. Villadangos, G. Dranoff, C. Small, L. Gu, K.J. Haley, R. Riese, H.L. Ploegh, H.A. Chapman, Immunity 10 (1999) 197–206. [4] G.-P. Shi, R.A. Bryant, R. Riese, S. Verhelst, C. Driessen, Z. Li, D. Bro¨mme, H.L. Ploegh, H.A. Chapman, J. Exp. Med. 191 (2000) 1177–1186. [5] C.T. Pham, T.J. Ley, Proc. Natl. Acad. Sci. USA 96 (1999) 8627– 8632. [6] C. Tepel, D. Bro¨mme, V. Herzog, C. Brix, J. Cell Sci. 113 (2000) 4487–4498.
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